Cotton transgenic event mon 88702 and methods for detection and uses thereof

ABSTRACT

The invention provides a transgenic  Gossypium hirsutum  event MON 88702, plants, plant cells, seeds, plant parts, progeny plants, and commodity products comprising event MON 88702. The invention also provides polynucleotides specific for event MON 88702, plants, plant cells, seeds, plant parts, progeny plants, and commodity products comprising polynucleotides for event MON 88702. The invention also provides methods related to event MON 88702.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/249,758, filed Nov. 2, 2015, which is herein incorporated byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file namedMONS405US-sequence_listing.txt is 33.3 kilobytes (size as measured inMicrosoft Windows®), was created on Oct. 25, 2016, is filed herewith byelectronic submission, and is incorporated by reference.

FIELD OF THE INVENTION

The invention relates to a transgenic cotton event referred to as MON88702. The event provides resistance from Hemipteran and Thysanopteraninfestations of cotton by providing a unique insecticidal toxin proteinnot previously available in cotton plants. This insecticidal toxinprotein is highly efficacious for controlling Hemipteran andThysanopteran species infestations characteristic to cotton plants. Theinvention also relates to cotton plants, plant parts, plant seeds, plantcells, progeny plants, agricultural products, and methods related toevent MON 88702, and provides nucleotide molecules that are unique tothe event, created in connection with the insertion of transgenic DNAinto the genome of a Gossypium hirsutum (cotton) cell, and useful fordetecting the presence of this event in biological samples containingcotton nucleic acids.

BACKGROUND OF THE INVENTION

Cotton is an important crop in many areas of the world, andbiotechnology methods have been applied to produce cotton varieties withdesirable traits. One such desirable trait is insect resistance. Theexpression of an insect resistance transgene in a cotton plant canconfer the desirable trait of insect resistance. Many different factorsinfluence the expression of a transgene, including the orientation andcomposition of the cassettes driving expression of the individual genestransferred to the plant chromosome, the chromosomal location of thetransgene insert, and the genomic result of the transgene insertion. Forexample, there can be variation in the level and pattern of transgeneexpression among individual events that differ in the chromosomalinsertion site of the transgene, but are otherwise identical. There canalso be phenotypic and agronomic differences between events.

To make a transgenic cotton plant containing a single transformationevent, a portion of a recombinant DNA construct is transferred into thegenome of a cotton cell, and the cotton cell is subsequently grown intoa plant. A cotton cell into which the event is initially transferred isregenerated to produce the R0 generation. The R0 plant and progenyplants from the R0 plant can be tested for any desired trait(s), but theeffectiveness of the event can be impacted by cis and/or trans factorsrelative to the integration site in the transformation event. Thephenotype conferred by the event can also be impacted by the size anddesign of the DNA construct, which can vary by the combination ofgenetic elements in an expression cassette, number of transgenes, numberof expression cassettes, and configuration of such elements and suchcassettes. Identifying an event with desirable traits can be furthercomplicated by factors such as plant developmental, diurnal, temporal,or spatial patterns of transgene expression, or by extrinsic factorssuch as environmental plant growth conditions, water availability,nitrogen availability, heat, or stress. Thus, the ability to obtain anevent conferring a desirable level of transgene expression and adesirable set of phenotypic and agronomic traits is not readilypredictable.

Due to these numerous factors that have an effect on the efficacy of anevent, it is necessary to produce and analyze a large number ofindividual plant cell transformation events in order to create an eventhaving proper expression of the desirable trait and optimal phenotypicand agricultural characteristics suitable for commercial success.Creating a commercially valuable transgenic event requires extensivemolecular characterization, as well as greenhouse and field trials withnumerous experimental events over multiple years, in multiple locations,and under a variety of conditions. A significant amount of efficacy,phenotypic, and molecular data is collected, and the resulting data andobservations are then analyzed by teams of scientists and agronomistswith the goal of selecting one or more commercially suitable events.Such an event, once selected, is then used for introgression of thedesirable transgenic trait into other genetic backgrounds using plantbreeding methods, thus producing a number of different cotton cropvarieties that contain the desirable trait and are suitably adapted tospecific local agronomic conditions.

Transgenic cotton plants are known in the art, but only plantsexpressing Lepidopteran toxins or herbicide tolerance genes have beenproduced. There are no commercial transgenic cotton plants for thecontrol of Hemipteran (such as lygus, cotton fleahopper and verde plantbug) and Thysanopteran (such as thrips) pests of cotton crops.

Lygus species can threaten a cotton crop from earliest squaring throughcutout and final boll set. The insects pierce squares and damage anthersand other tissues. When squares are less than 5 millimeters long, theyshrivel, turn brown, and drop from the plant. Damage to larger squaresmay be to anthers, styles, and stigma, and may interfere withfertilization. If many squares drop, the plant may put its energyresources into vegetative growth, resulting in tall, spindly plants withreduced yields. Lygus species also feed on and destroy terminalmeristems, causing bushy plants. If lygus pierce the wall of young bolls(typically less than ten days old) and feed on young seeds, these seedsmay fail to develop. Lint around the injured seeds is stained yellow,and may not mature normally.

Cotton fleahopper can cause excessive loss of cotton squares, resultingin reduced yield and harvest delays. Cotton fleahopper is a key insectpest of cotton in Texas and Oklahoma, and an occasional pest in NewMexico, Arkansas, Louisiana, and other mid-South states of the UnitedStates of America. When heavy populations of the insect pest are leftuncontrolled, the yield loss can become extremely high. Cottonfleahopper nymphs and adults feed on the juices of tender plant parts,especially the terminal buds and small squares. Deformed or raggedleaves are often seen as a result of this feeding. The greatest damageis to small squares that are no larger than a pinhead. The small squaresturn brown or black and shed after being fed upon. Heavily infestedplants grow tall and whip-like, have restricted growth of fruitingbranches, and usually produce only a few bolls near the top. The insectand its damage is thus hard to detect until economic losses have beensustained.

Verde plant bug, a native species, emerged as an important boll-feederalong the Gulf Coast of the United States of America during the past tento fifteen years. During this time, cotton yields have suffered lossesfrom cotton boll rot in areas of South Texas. Piercing-sucking insectsfeeding on cotton bolls have been implicated in introducing thebacterial disease that causes boll rot. Verde plant bug was the dominantboll-feeding sucking bug species (>98% of insects collected using a beatbucket) from peak to late bloom in cotton fields near the coast alongthe Coastal Bend of South Texas, from Port Lavaca to the Lower RioGrande Valley in 2010 and 2011. It was common in fields within 8 km ofcoastal waters (average of 0.42 bugs per plant during peak to latebloom), while it was not detected in inland fields. Cotton boll rot wasfound on up to 25% of the open bolls inspected, the disease wasconcentrated in coastal fields where verde plant bug was found, and itwas the major contributor to boll damage. Results from field surveys andverde plant bug feeding on caged plants supported the positiveassociation of verde plant bug presence and subsequent harvest-relevantcotton boll rot in open bolls at harvest (Armstrong, J., Brewer, M.,Parker, R., and Adamzyk, J. (2013) Verde Plant Bug (Hemiptera: Miridae)Feeding Injury to Cotton Bolls Characterized by Boll Age, Size, andDamage Ratings, J. Econ. Entomol. 106(1): 189D195). Verde plant bugfeeding on cotton bolls also results in lint and seed staining.

Thrips have “punch and suck” mouthparts that allow them to punch a holein a leaf cell, insert their maxillary stylets, and suck up the cellularfluids. When thrips feed on terminal buds, on tiny developing leaves andon fruiting structures, the injury can be severe. When thrips feed onyoung undeveloped leaves within the terminal bud, the resulting damageis magnified as those leaves develop and expand. This is because thedamaged tissue fails to develop properly, while undamaged tissuecontinues to grow. After prolonged feeding or feeding by high numbers ofthrips, seedlings have a ragged appearance, with visible silvery feedingsites on cotyledons and terminal leaf tissue. Over time, these silverareas will turn brown in color. Heavily injured leaves usually have acrinkled, tattered appearance and often curl upwards at the margins.Seedlings with this type of injury are often described as “possum-earedcotton.” Heavy thrips populations can stunt growth, cause death of theterminal bud (resulting in “crazy cotton”), delay fruiting, and reducestand. Severe thrips injury can result in substantial cotton yieldreductions. Both larvae and adults show a preference to feed on and inflowers, making them particularly difficult to control with chemicalpesticides. In addition, Frankliniella occidentalis (Western flowerthrips) is a very efficient vector of different plant topoviruses (e.g.,tomato spotted wilt virus and Cotton leaf roll virus) that cause damageto cotton plants. In sum, thrips can severely impact early season standand plant vigor and can be very costly to cotton farmer.

Chemical pesticides are the current insect control methods used againstLygus hesperus (Western tarnished plant bug), Lygus lineolaris(Tarnished plant bug), Pseudatomoscelis seriatus (Cotton fleahopper),Creontiades signatus Distant (Verde plant bug), and the thysanopteranpests Frankliniella spp and Sericothrips variabilis (Thrips). Thesechemical insecticide methodologies often require multiple applicationsand different types of chemical pesticides. For example, to controllygus as many as five to ten treatments per season may be required. Tocontrol thrips, two to three treatments per season may be required. Atleast one treatment is required to control cotton fleahopper.

The use of chemical pesticides to control insect pests increases thecost to the farmer growing cotton, particularly in regions experiencinghigh insect infestation, thus reducing any potential profit derived fromcotton production. In addition, the use of different chemical pesticideswith multiple applications can have a negative impact on the environmentand beneficial insects. Further, the development of resistance tochemical pesticides has been observed in these cotton pests. Forexample, the western flower thrips, Frankliniella occidentalis Pergande,has shown resistance to a number of different chemical pesticides (StenJensen (2006) Insecticide resistance in the western flower thrips,Frankliniella occidentalis, Integrated Pest Management Reviews, 5:131-146). Resistance to multiple classes of chemical pesticides(carbamate, organophosphate, and pyrethroid insecticides) used tocontrol Tarnished plant bug (Lygus lineolaris) has also been observed(GL Snodgrass et al., (2009) Acephate resistance in populations of thetarnished plant bug (Heteroptera: Miridae) from the Mississippi RiverDelta. J Econ Entomol, 102(2): 699-707).

Because of the economic and environmental cost associated with the useof chemical pesticides to control insect pests of cotton, and thedevelopment of resistance to chemical pesticides, there is a need for acotton plant that expresses an insecticidal toxin active against Lygushesperus (Western tarnished plant bug), Lygus lineolaris (Tarnishedplant bug), Pseudatomoscelis seriatus (Cotton fleahopper), Creontiadessignatus (Distant) (Verde plant bug), and the thysanopteran pestsFrankliniella spp and Sericothrips variabilis (Thrips).

SUMMARY OF THE INVENTION

In one aspect, the invention provides transgenic cotton plantscomprising event MON 88702 exhibiting superior properties andperformance compared to existing transgenic cotton plants and to newevents constructed in parallel. The cotton event MON 88702 contains, ata single locus of insertion in the cotton genome, an expression cassettewhich confers the trait of resistance to Hemipteran and Thysanopteraninsect pests.

In one embodiment, event MON 88702 is characterized by specific uniqueDNA segments that are useful in detecting the presence of the event in asample. A sample is intended to refer to a composition that is eithersubstantially pure cotton DNA or a composition that contains cotton DNA.In either case, the sample is a biological sample, i.e., it containsbiological materials, including but not limited to DNA obtained orderived from, either directly or indirectly, the genome of cottoncomprising event MON 88702. “Directly” refers to the ability of theskilled artisan to directly obtain DNA from the cotton genome byfracturing cotton cells (or by obtaining samples of cotton that containfractured cotton cells) and exposing the genomic DNA for the purposes ofdetection. “Indirectly” refers to the ability of the skilled artisan toobtain the target or specific reference DNA, i.e. a novel and uniquejunction segment described herein as being diagnostic for the presenceof the event MON 88702 in a particular sample, by means other than bydirect via fracturing of cotton cells or obtaining a sample of cottonthat contains fractured cotton cells. Such indirect means include, butare not limited to, amplification of a DNA segment that contains the DNAsequence targeted by a particular probe designed to bind withspecificity to the target sequence, or amplification of a DNA segmentthat can be measured and characterized, i.e. measured by separation fromother segments of DNA through some efficient matrix such as an agaroseor acrylamide gel or the like, or characterized by direct sequenceanalysis of the amplicons, or cloning of the amplicon into a vector anddirect sequencing of the inserted amplicon present within such vector.Alternatively, a segment of DNA corresponding to the position within thecotton chromosome at which the transgenic DNA was inserted into thecotton chromosome and which can be used to define the event MON 88702,can be cloned by various means and then identified and characterized forits presence in a particular sample or in a particular cotton genome.Such DNA segments are referred to as junction segments or sequences, andcan be any length of inserted DNA and adjacent (flanking) cottonchromosome DNA so long as the point of joining between the inserted DNAand the cotton genome is included in the segment. In certainembodiments, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, and SEQ ID NO:10 and the reverse complement of eachof these, are representative of such segments.

The specific sequences identified herein may be present uniquely inevent MON 88702, or the construct comprised therein, and theidentification of these sequences, whether by direct sequence analysis,by detecting probes bound to such sequences, or by observing the sizeand perhaps the composition of particular amplicons described herein,when present in a particular cotton germplasm or genome and/or presentin a particular biological sample containing cotton DNA, are diagnosticfor the presence of the event MON 88702, or the construct comprisedtherein, in such sample. It is known that the flanking genomic segments(i.e., the cotton genome segments of DNA sequence adjacent to theinserted transgenic DNA) are subject to slight variability and as such,the limitation of at least 99% or greater identity is with reference tosuch anomalies or polymorphisms from cotton genome to cotton genome. Inone embodiment, nucleotide segments that are completely complementaryacross their length in comparison to the particular diagnostic sequencesreferenced herein are intended to be within the scope of the presentinvention.

The position of the nucleotide segments of the present inventionrelative to each other and within the cotton genome are illustrated inFIG. 1 and the nucleotide sequence of each is illustrated as set forthin SEQ ID NO:10. Nucleotide segments that characterize the event MON88702 and which are diagnostic for the presence of event MON 88702, orthe construct comprised therein, in a sample include SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, orSEQ ID NO:26. The presence of one, or two, or more of these nucleotidesequences in a sample, when such sample contains cotton tissue and thuscotton DNA, is diagnostic for the presence of the event MON 88702, orthe construct comprised therein.

It is intended by use of the word “derived” that a particular DNAmolecule is in the cotton plant genome, or is capable of being detectedin cotton plant DNA. “Capable of being detected” refers to the abilityof a particular DNA segment to be amplified and its size and or sequencecharacterized or elucidated by DNA sequence analysis, and can also referto the ability of a probe to bind specifically to the particular DNAsegment, i.e. the target DNA segment, and the subsequent ability todetect the binding of the probe to the target. In certain embodiments,the particular DNA segment or target DNA segment of the presentinvention is present within cotton that contains the insertion event MON88702.

By reference to cotton it is intended that cotton cells, cotton seed,cotton plant parts and cotton plants are within the scope of the presentinvention so long as each embodiment contains a detectable amount of DNAcorresponding to any one, two, or more of the segments that aredescribed herein as being diagnostic for the presence of the cottonevent MON 88702 DNA. In certain embodiments, cotton plant parts includecells; pollen; ovules; flowers; lint; seed; root tissue; stem tissue;and leaf tissue. Commodity products that are made from cotton in which adetectable amount of the segments of DNA described herein as beingdiagnostic for the presence of the event MON 88702 are within the scopeof the invention. In some embodiments, such commodity products mayinclude whole or process cotton seeds, cotton fiber, cotton oil andderivatives of cotton oil, cotton protein, cotton meal, animal feedcomprising cotton, paper comprising cotton, cotton biomass, candlewicks, cotton string, cotton rope, cotton balls, cotton batting, cottonfuel products such as fuel derived from cotton oil or pellets derivedfrom cotton gin waste, and cotton cellulose products such as rayon,plastics, photographic film, cellophane, fatty acids used for variousindustrial uses such as insulation materials, linoleum, oilcloth,waterproofing, and as a paint base.

In one embodiment, the DNA of cotton event MON 88702 may be present ineach cell and in each genome on one chromosome of the cotton plant,cotton seed, and cotton tissues containing the event. As the cottongenome is transmitted to progeny in Mendelian fashion, if a cotton plantwere homozygous for the event MON 88702 insertion, each progeny cottonplant and cell would contain the event DNA on each allele of theparental chromosome containing the event MON 88702 insertion andinherited by the progeny from the parent(s). However, if the cottongenome containing the event MON 88702 DNA is a heterozygous or hybridparent, then about fifty percent of the pollen and about fifty percentof the ovules engaged in mating from hybrid parents will contain thecotton event MON 88702 DNA, resulting in a mixed population of progenythat contain the event MON 88702 DNA, and the percentage of such progenyarising from such crosses with hybrids can range anywhere from aboutfifty to about seventy five percent having the event MON 88702 DNAtransmitted to such progeny.

In another embodiment, the DNA molecules of the present invention may beunique to the two separate junctions on either end of the insertedtransgenic event MON 88702 DNA and the cotton genome DNA that isadjacent to, i.e. flanking, each end of the MON 88702 inserted DNA, orunique to the cotton event MON 88702 inserted DNA. DNA molecules havingthe sequence of these junction sequences, when present in a particularsample of cotton analyzed by the methods described herein using theprobes, primers and in some cases using DNA sequence analysis, may bediagnostic for the presence of an amount of event MON 88702 cotton inthat sample. Such DNA molecules unique to the cotton event MON 88702 DNAcan be identified and characterized in a number of ways, including byuse of probe nucleic acid molecules designed to bind specifically to theunique DNA molecules followed by detection of the binding of such probesto the unique DNA, and by thermal amplification methods that use atleast two different DNA molecules that act as probes but the sequence ofsuch molecules may be somewhat less specific than the probes describedabove. The skilled artisan understands that contacting a particulartarget DNA with a probe or primer under appropriate hybridizationconditions will result in the binding of the probe or primer to thetargeted DNA segment.

In one embodiment, the DNA molecules of the present invention may betarget segments of DNA that may be capable of amplification and, whendetected as one or more amplicons of the represented length obtained byamplification methods of a particular sample, may be diagnostic for thepresence of event MON 88702, or the construct comprised therein, in suchsample. In another embodiment, such DNA molecules or polynucleotidesegments may have the nucleotide sequences as set forth in each of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, and SEQ ID NO:26, and are further defined hereinand in the examples below. Primer molecules and/or probes may beprovided in kit form along with the necessary reagents, includingcontrols, and packaged together with instructions for use.

In one aspect, recombinant DNA molecules of the present invention aredeemed to be within the scope of the present invention when presentwithin or derived from a microorganism. In one embodiment, amicroorganism is intended to include any microscopic cell, whetherprokaryote or eukaryote or otherwise that contains DNA within a genomeor chromosome or an extra-chromosomal DNA structure more commonlyreferred to as a plasmid or vector. In other embodiments, microscopicorganisms include bacteria (prokaryotes) and cells corresponding tohigher life forms (eukaryotes) which are beneath the visual range of theaverage human, typically beneath fifty cubic microns and more generallybeneath ten cubic microns. Bacteria are common microscopicmicroorganisms that more likely than not could contain a vector orplasmid that contains one or more or all of the novel DNA segments ofthe present invention, including the expression cassette present as setforth in SEQ ID NO:27. In other aspects, plant cells and particularlycotton plant cells are within the scope of the invention when thesecontain any one, two, or more or all of the novel DNA segments of thepresent invention.

In another aspect, probes for use herein may comprise DNA molecules orpolynucleotide segments of sufficient length to function under stringenthybridization conditions as defined herein to bind with a particulartarget DNA segment, i.e., a unique segment of DNA present within anddiagnostic for the presence of, event MON 88702 DNA in a sample. Such aprobe can be designed to bind only to a single junction or other novelsequence present only in the cotton event MON 88702 DNA, or to two ormore such single junction segments. In one embodiment, the detection ofthe binding of such a probe to a DNA molecule in a particular samplesuspected of containing cotton DNA is diagnostic for the presence ofcotton event MON 88702 in the sample.

In yet another aspect, primers may comprise pairs of differentoligonucleotides or polynucleotide segments for use in a thermalamplification reaction which amplifies a particular DNA target segment.Each primer in the pair is designed to bind to a rather specific segmentof DNA within or near to a segment of DNA of interest for amplification.The primers bind in such way that these then act as localized regions ofnucleic acid sequence polymerization resulting in the production of oneor more amplicons (amplified target segments of DNA). In one embodimentof the present invention, use of primers designed to bind to uniquesegments of cotton event MON 88702 DNA in a particular biological sampleand that amplify particular amplicons containing one or more of thejunction segments described herein, and the detection and/orcharacterization of such amplicons upon completion or termination of thepolymerase reaction, is diagnostic for the presence of the cotton eventMON 88702 in the particular sample. The skilled artisan is well familiarwith this amplification method and no recitation of the specifics ofamplification is necessary here.

In a further aspect, the present invention provides cotton plants,cotton plant cells, cotton plant tissues and cotton seed resistant toinfestation by Hemipteran and Thysanopteran insect pests, including butnot limited to Lygus hesperus, Lygus lineolaris, Pseudatomoscelisseriatus, Creontiades signatus, Frankliniella spp, and Sericothripsvariabilis. In a particular embodiment, the resistance to infestation byHemipteran and Thysanopteran species arises in connection with theexpression of a DNA segment, encoding an insecticidal protein, that isoperably and covalently linked within the inserted transgenic DNA: aTIC834_16 protein (United States Patent Application, US20130269060, SEQID NO:34) expressed from the expression cassette within the insertedtransgenic DNA as set forth in SEQ ID NO:10 and illustrated in FIG. 1 b.

The TIC834_16 protein which provides resistance to Hemipteran andThysanopteran insect pests in cotton event MON 88702 is expressed by achimeric FMV/Hsp81.2 chimeric promoter (U.S. Pat. No. 8,940,962). FIG.1b shows the relative position of each expression element (enhancer (E),promoter (P), 5′ UTR (L), 3′ UTR (T)) and the TIC834_16 coding sequencewithin the transgene cassette comprised within SEQ ID NO:10. Otherconstructs were evaluated and varied in the use expression elements. Thetransgene cassette used to create cotton event MON 88702 providedsuperior performance to other transgene cassettes when evaluated forresistance to Hemipteran and Thysanopteran insect pest infestation.

The event MON 88702 was selected based on comparisons to thousands ofdifferent independent transgenic events, each transformed with aconstruct comprising the transgene cassette presented as SEQ ID NO:27 orother constructs comprising the TIC834_16 coding sequence or relatedvariant toxin proteins expressed using different expression elements.The events generated expressing TIC834_16 and variants related toTIC834_16 were compared to the nontransgenic control cotton varietyDP393 for resistance to Hemipteran and Thysanopteran insect pests. Theresults as illustrated in the Examples below show that the event MON88702 yields superior properties due to expression of the TIC834_16protein. The plurality of transgenic events produced using the constructused for generating the event MON 88702 were each more likely than otherevents produced with other constructs to exhibit efficacious control ofHemipteran and Thysanopteran insect pests.

In one aspect, cotton plants and parts thereof including seed, eachcontaining the DNA corresponding to event MON 88702, are within thescope of the present invention. In one embodiment, such plants and partsthereof including seed are resistant to Hemipteran and Thysanopteraninfestation. In certain embodiments, such plants and seed includehybrids and inbreds, and plants and seed that contain only one event MON88702 allele, i.e., a genome characterized as heterozygous withreference to the locus corresponding to the event MON 88702 DNA. Suchhybrids may be produced by breeding plants comprising event MON 88702with desirable germplasm as part of the commercial variety developmentprocess and other agriculturally desirable properties of cotton. Hybridsmay be produced by any number of methods but a preferred method takesadvantage of a first inbred (homozygous) parent that contains the eventMON 88702 specific allele on both chromosomes at the locus at which theevent MON 88702 DNA is inserted, and breeding the first inbred togetherwith a second inbred which does not contain the MON 87702 DNA. Bothparental inbred varieties will have one or more advantageous propertiesdesirable in the progeny seed, i.e. the hybrid seed, and these hybridseed are heterozygous for the event MON 88702 allele.

In one embodiment, a transgenic property or allele conferring someadditional trait to a plant containing the event MON 88702 DNA may bedesirable. In another emodiment, other such transgenic allelesconferring desirable traits may include herbicide tolerance: 19-51A(DD-04951A-7), BXN, MON 1445 (MON-01445-2), MON 88701 (MON-88701-3), MON88913 (MON-88913-8), GHB614 (BCS-GH002-5), DAS-81910-7 (DAS-81910-7),GHB119 (BCS-GH005-8), LLCotton25 (ACS-GH0013), EE-GH1, EE-GH3,pDAB4468.18.07.1, and pDAB4468.19.10.3; insect resistance: 281-24-236(DAS-24236-5), 3006-210-23 (DAS-21023-5), COT102 (SYN-IR102-7), COT67B(SYN-IR67B-1), Event-1, MON 531 (MON-00531-6), MON15985 (MON-15985-7),EE-GHS, EE-GH6, COT202, COT203, and A26-5; and insect resistance andherbicide tolerance: 31807, 31808, T303-3 (BCS-GH003-6) and T304-40(BCS-GH004-7). A non-transgenic property (e.g., QTL or maturity group)may also confer a desirable trait and one with skill in the art wouldknow how to breed cotton to contain such non-transgenic trait and eventMON 88702 DNA. Certain varieties of cotton have properties that may bedesirable to further reduce damage from insect pests and may be suitableas a parent line in breeding cotton that also comprises event MON 88702DNA. Early maturing, short season varieties are more likely to escapeattack and damage from late-season infestations of budworms, bollwormsand lygus. Smooth leaf varieties tend to have reduced populations ofaphid and whitefly populations while budworms and bollworms depositfewer eggs when compared to hairy varieties. Okra leaf varieties withokra leaf trait allow improved canopy penetration of foliar insecticidetreatments and the trait also has been associated with resistance towhiteflies. Nectariless varieties tend to have lower plant bugpopulations and reduced egg production capacity of most moth speciesbecause of reduced nectar availability. High glanding varieties withhigh glanding trait have additional gossypol glands which increases theresistant to budworms and bollworms.

The forgoing and other aspects of the invention will become moreapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a diagrammatic representation of the relative positionsillustrated by each horizontal line, of the segments of the heterologoustransgenic DNA, the flanking genomic DNA, the arbitrarily designated 5′and 3′ genomic/inserted DNA junctions, and relative positions ofsequence unique to event MON 88702 within the heterologous transgenomicDNA which may be used to identify cotton event MON 88702; the horizontallines labeled [1], [2], [3], [4], [5], [6], [7], [8], [9], and [10]correspond to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ IDNO:10, respectively; the horizontal bar labeled [10] (SEQ ID NO:10)represents the contig of the 5′ genomic flanking DNA sequence ([7], SEQID NO:7), the inserted T-DNA cassette ([9], SEQ ID NO:9), and the 3′flanking DNA sequence ([8], SEQ ID NO:8); the small horizontal lineslabeled [1] (SEQ ID NO:1), [2] (SEQ ID NO:2), [3] (SEQ ID NO 3), [4](SEQ ID NO:4), [5] (SEQ ID NO:5), and [6] (SEQ ID NO:6) represent uniquesequences of the genomic/insert DNA junctions that can be used toidentify the presence of event MON 88702 in a biological sample; thethick arrows labeled [11] SQ21940 (SEQ ID NO:11), [12] SQ-50210 (SEQ IDNO:12), [17] SQ50844 (SEQ ID NO:17), [19] SQ50843 represent a subset ofprimers used in the event specific assay and the zygosity assay, and arepositioned relative to where they hybridize to SEQ ID NO:10; the thickarrows labeled [13] PB10344 and [18] PB50279 are a subset of probes usedin the event specific assay and the zygosity assay, and are positionedrelative to where they hybridize to SEQ ID NO:10.

FIG. 1b is a diagrammatic representation of the T-DNA cassette in theplasmid vector used to transform event MON 88702, presented as SEQ IDNO:27 ([27]); the horizontal arrows below [27] represents the elementscomprised within the T-DNA cassette (SEQ ID NO: 27); RB represents aT-DNA right border element, E represents an enhancer element, Prepresents a promoter element, L represents a 5′ UTR, TIC834_6represents the TIC834_6 coding sequence element, T represents a 3′ UTR,and LB represents a T-DNA left border element; the horizontal lineslabeled [22], [23], [24], [25], and [26] correspond to SEQ ID NO:22, SEQID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively andrepresent unique sequences of the junctions between elements or thecoding sequence which can be used to identify the presence of event MON88702 in a biological sample.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a twenty nucleotide sequence representing the 5′ junctionregions of cotton genomic DNA and the integrated transgenic expressioncassette. SEQ ID NO:1 is found within SEQ ID NO:10 at nucleotideposition 1642-1661.

SEQ ID NO:2 is a twenty nucleotide sequence representing the 3′ junctionregions of cotton genomic DNA and the integrated transgenic expressioncassette. SEQ ID NO:2 is found within SEQ ID NO:10 at nucleotideposition 4785-4804.

SEQ ID NO:3 is a sixty nucleotide sequence representing the 5′ junctionregions of cotton genomic DNA and the integrated transgenic expressioncassette. SEQ ID NO:3 is found within SEQ ID NO:10 at nucleotideposition 1622-1681.

SEQ ID NO:4 is a sixty nucleotide sequence representing the 3′ junctionregions of cotton genomic DNA and the integrated transgenic expressioncassette. SEQ ID NO:4 is found within SEQ ID NO:10 at nucleotideposition 4765-4824.

SEQ ID NO:5 is a one hundred nucleotide sequence representing the 5′junction regions of cotton genomic DNA and the integrated transgenicexpression cassette. SEQ ID NO:5 is found within SEQ ID NO:10 atnucleotide position 1602-1701.

SEQ ID NO:6 is a one hundred nucleotide sequence representing the 3′junction regions of cotton genomic DNA and the integrated transgenicexpression cassette. SEQ ID NO:6 is found within SEQ ID NO:10 atnucleotide position 1602-1701.

SEQ ID NO:7 is a 1,651 nucleotide sequence representing the 5′ flankingcotton genomic sequence up to the inserted T-DNA.

SEQ ID NO:8 is a 2,010 nucleotide sequence representing the 3′ flankingcotton genomic sequence after the inserted T-DNA. The first fournucleotides of SEQ ID NO:8 are derived from an unknown origin and arenot present in the non-transgenic DP393 variety which was used totransform cotton event MON 88702; and were likely introduced during theintegration of the MON 88702 T-DNA.

SEQ ID NO:9 is a 3,143 nucleotide sequence corresponding to thetransgenic inserted T-DNA of cotton event MON 88702.

SEQ ID NO:10 is a 6,804 nucleotide sequence corresponding to the contignucleotide sequence of the 5′ genomic flanking DNA nucleotide sequence,the inserted T-DNA nucleotide sequence in event MON 88702, and the 3′genomic flanking DNA nucleotide sequence; and includes SEQ ID NO:7(nucleotides 1-1651), SEQ ID NO:9 (nucleotides 1652-4794), and SEQ IDNO:8 (nucleotides 4795-6804).

SEQ ID NO:11 is a 25 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ21940 used to identify cottonevent MON 88702 DNA in a sample, and is identical to the nucleotidesequence corresponding to positions 4720 to 4744 of SEQ ID NO:10.

SEQ ID NO:12 is a 24 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ-50210 used to identify cottonevent MON 88702 DNA in a sample, and is identical to the reversecompliment of the nucleotide sequence corresponding to positions 4803 to4826 of SEQ ID NO:10.

SEQ ID NO:13 is a 22 nucleotide sequence corresponding to a probereferred to as PB10344 used to identify cotton event MON 88702 DNA in asample, and is identical to the nucleotide sequence corresponding topositions 4745 to 4766 of SEQ ID NO:10.

SEQ ID NO:14 is a 23 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ22496 used as an internal controlin the event assay for MON 88702 and hybridizes to the Gossypiumhirsutum fiber-specific acyl carrier protein (ACP1) gene (GenBankAccession U48777).

SEQ ID NO:15 is a 19 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ22497 used as an internal controlin the event assay for MON 88702 and hybridizes to the complimentarystrand of the Gossypium hirsutum fiber-specific acyl carrier protein(ACP1) gene (GenBank Accession U48777).

SEQ ID NO:16 is a 20 nucleotide sequence corresponding to a probereferred to as PB13032 used as an internal control in the event assayfor MON 88702 and hybridizes to the Gossypium hirsutum fiber-specificacyl carrier protein (ACP1) gene (GenBank Accession U48777).

SEQ ID NO:17 is a 24 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ50844 used in the zygosity assayfor event MON 88702 DNA in a sample, and is identical to the nucleotidesequence corresponding to positions 4672 to 4695 of SEQ ID NO:10.

SEQ ID NO:18 is a 20 nucleotide sequence corresponding to a probereferred to as PB50279 used to identify the cotton event MON 88702allele in a sample for the zygosity assay, and is identical to thenucleotide sequence corresponding to positions 4735 to 4754 of SEQ IDNO:10.

SEQ ID NO:19 is a 26 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ50843 used in the zygosity assayfor event MON 88702 DNA in a sample which hybridizes to both thewild-type and MON 88702 alleles, and is identical to the reversecompliment of the nucleotide sequence corresponding to positions 4852 to4877 of SEQ ID NO:10.

SEQ ID NO:20 is a 25 nucleotide sequence corresponding to a thermalamplification primer referred to as SQ50842 used in the zygosity assayfor event MON 88702 DNA in a sample which hybridizes to only thewild-type DNA allele that does not contain the inserted MON 88702transgene cassette in a region of wild-type DNA that was lost duringtransgene insertion and therefore does not correspond to a region alongSEQ ID NO:10.

SEQ ID NO:21 is a 21 nucleotide sequence corresponding to a probereferred to as PB50278 used to identify the wild-type allele in thezygosity assay for event MON 88702 DNA in a sample, and hybridizes toonly the wild-type DNA allele that does not contain the inserted MON88702 transgene cassette in a region of wild-type DNA that was lostduring transgene insertion and therefore does not correspond to a regionalong SEQ ID NO:10.

SEQ ID NO:22 is a 101 nucleotide sequence representing the junctionbetween the right T-DNA border sequence and the FMV 35S enhancersequence within the transgene cassette used to transform DP393 cotton toproduce cotton event MON 88702 which includes 15 nucleotides of theright T-DNA border sequence, 53 nucleotides of intervening sequence, and33 nucleotides of the enhancer sequence. SEQ ID NO:22 is positioned inSEQ ID NO:10 at nucleotide position 1705-1805.

SEQ ID NO:23 is a 77 nucleotide sequence representing the junctionbetween the Arabidopsis thaliana HSP81.2 5′ UTR and the TIC834_16 codingsequence within the transgene cassette used to transform DP393 cotton toproduce cotton event MON 88702 which includes 19 nucleotides of the 5′UTR, 37 nucleotides of intervening sequence, and 21 nucleotides of theTIC834_16 coding sequence. SEQ ID NO:23 is positioned in SEQ ID NO:10 atnucleotide position 3244-3320.

SEQ ID NO:24 is a 97 nucleotide sequence representing the junctionbetween the TIC834_16 coding sequence and the 3′ UTR within thetransgene cassette used to transform DP393 cotton to produce cottonevent MON 88702 which includes 28 nucleotides of the TIC834_16 codingsequence, 32 nucleotides of intervening sequence, and 37 nucleotides of3′ UTR sequence. SEQ ID NO:24 is positioned in SEQ ID NO:10 atnucleotide position 4193-4289.

SEQ ID NO:25 is a 200 nucleotide sequence representing the junctionbetween the 3′ UTR and the left T-DNA border sequence within thetransgene cassette used to transform DP393 cotton to produce cottonevent MON 88702 which includes 29 nucleotides of the 3′ UTR sequence,138 nucleotides of intervening sequence, and 33 nucleotides of the leftT-DNA border sequence. SEQ ID NO:25 is positioned in SEQ ID NO:10 atnucleotide position 4424-4623.

SEQ ID NO:26 is a 921 nucleotide sequence representing the TIC834_16coding sequence within the transgene cassette used to transform DP393cotton to produce cotton event MON 88702. SEQ ID NO:26 is positionedwithin SEQ ID NO:10 at nucleotide position 3300-4220.

SEQ ID NO:27 is a 3,598 nucleotide sequence representing the transgenecassette comprised within the binary plasmid transformation vector usedto transform DP393 cotton to produce cotton event MON 88702. SEQ ID NO:9the 3143 nucleotide sequence corresponding to the transgenic insertedDNA of cotton event MON 88702 is positioned in SEQ ID NO:27 atnucleotide position 254-3360.

DETAILED DESCRIPTION

The present invention provides a transgenic cotton plant that achievesinsecticidal control over Hemipteran and Thysanopteran pests of cottonby expression of TIC834_16, i.e., MON 88702. Specifically, expression ofthe TIC834_16 insect inhibitory protein in cotton event MON 88702provides resistance to the Hemipteran pests Lygus hesperus (Westerntarnished plant bug), Lygus lineolaris (Tarnished plant bug),Pseudatomoscelis seriatus (Cotton fleahopper), Creontiades signatusDistant (Verde plant bug), and Thysanoptera such as Frankliniella sppand Sericothrips variabilis (Thrips). The event MON 88702 will meet agreat need for control of these insects in the cotton market, aschemical insecticides often do not provide adequate control of theseinsects, or require multiple applications over the growing season,increasing the input of chemical pesticides in the environment andadding cost to the production of cotton.

Cotton event MON 88702 was produced by an Agrobacterium-mediatedtransformation process of cotton meristem tissue with a two T-DNA binarysystem. In this system, an Agrobacterium strain employing one binaryplasmid vector with two T-DNA transgene cassettes was utilized. Thefirst T-DNA transgene cassette was used for the selection of transformedcotton plant cells using a selectable marker which confers tolerance tothe antibiotic spectinomycin. The second transgene cassette was theTIC834_16 expression cassette—presented as SEQ ID NO: 27 herein—whichconfers resistance to Hemipteran and Thysanopteran insect pests. Theselection T-DNA cassette inserted randomly into the cotton genome at asite separate from the site of integration of the T-DNA containing theTIC834_16 expression cassette, thus allowing for segregation of the twoT-DNA segments within the genome of the transformed cotton plants duringthe process of selfing or backcrossing, e.g. screening R₁ and highergeneration transgenic plants.

The cotton cells transformed through this two T-DNA binary system wereregenerated into intact cotton plants. Individual plants that showedintegrity of the T-DNA cassette encoding the TIC834_16 protein, theabsence (i.e., segregation) of the T-DNA selectable marker cassette, andthe absence of plasmid backbone sequence were selected for furthertesting.

Specifically, over 200 transgenic events were produced using thetransformation construct used to produce the transgenic cotton event MON88702, and thirty eight additional constructs comprising TIC834_16 orrelated variants of TIC834_16 were generated and used to produce overtwo thousand one hundred eighty (2,180) other transgenic cotton eventswhich were compared to the cotton event MON 88702 and similar cottonevents. Many of these events were tested by ELISA assay for expressionin the leaf tissue of the insecticidal protein, TIC834_16 or relatedvariants. Many of these same events were examined for the presence ofprotein crystals which were the result of over-expression of the toxinsand interfered with efficacy. A subset of the events produced from eachtransformation, and most of the constructs, were tested for efficacy forcontrolling Hemipteran insect pests in cage and field trials. It wasdetermined that the plant expression elements in the construct used toproduce cotton event MON 88702 provided the events with the bestefficacy against the Hemipteran pests tested. Of the events created andtested, event MON 88702 was ultimately selected as the superior event inview of the beneficial and superior properties yielded thereby.

The plasmid DNA inserted into the genome of cotton event MON 88702 wascharacterized by detailed molecular analysis. This analysis included:the insert number (number of integration sites within the cottongenome), the genomic insert location (the specific site in the cottongenome where the insertion occurred), the copy number (the number ofcopies of the T-DNA within one locus), and the integrity of thetransgenic inserted DNA. The detailed molecular analysis demonstratedthat the plasmid construct containing the TIC834_16 expression cassettecontains multiple segments (junction sequences between elements used tobuild or construct the expression cassette). These segments (e.g.,sequences as set forth in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQID NO:25, and SEQ ID NO:26) are not known to appear naturally in thecotton genome or in other vectors or transgenic cotton events; they areunique to the event MON 88702. Further, cotton event MON 88702 ischaracterized as an insertion into a single locus in the cotton genome,resulting in two new loci or junction sequences (e.g., sequences as setforth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, and SEQ ID NO: 6) between the inserted DNA and the cotton genomeDNA that are not known to appear naturally in the cotton genome or inother transgenic cotton events; they are unique to the event MON 88702.These junction sequences are useful in detecting the presence of theevent MON 88702 in cotton cells, cotton tissue, cotton seed, and cottonplants or cotton plant products, such as cotton commodity products. DNAmolecular probes and primer pairs are described herein that have beendeveloped for use in identifying the presence of these various junctionsegments in biological samples containing or suspected of containingcotton cells, cotton seed, cotton plant parts or cotton plant tissuethat contain the event MON 88702.

The detailed molecular analysis also demonstrated that event MON 88702contains a single T-DNA insertion with one copy of the TIC834_16expression cassette. No additional elements from the transformationconstruct other than portions of the Agrobacterium tumefaciens right andleft border regions used for transgenic DNA transfer from the planttransformation plasmid to the cotton genome were identified in event MON88702. Finally, thermal amplification producing specific ampliconsdiagnostic for the presence of event MON 88702 in a sample and DNAsequence analyses were performed to determine the arbitrarily assigned5′ and 3′ insert-to-plant genome junctions, confirm the organization ofthe elements within the insert, and determine the complete DNA sequenceof the inserted transgenic DNA (SEQ ID NO:9) in cotton event MON 88702.SEQ ID NO:7 is a sequence representing the 5′ DP393 cotton genomic DNAsequence flanking the inserted T-DNA sequence presented as SEQ ID NO:9.SEQ ID NO:8 is a sequence representing the 5′ DP393 cotton genomic DNAsequence flanking the inserted T-DNA sequence presented as SEQ ID NO:9.SEQ ID NO:10 represents a contiguous sequence (contig) comprising the 5′DP393 flanking sequence, the inserted MON 88702 T-DNA and the 3′ DP393flanking sequence, and thus contains the insert-to-plant genome junctionsequences.

Unless otherwise noted herein, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art.Definitions of common terms in molecular biology may also be found inRieger et al., Glossary of Genetics: Classical and Molecular, 5^(th)edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, OxfordUniversity Press: New York, 1994. As used herein, the term “cotton”means species belong to the genus Gossypium, preferably Gossypiumhirsutum L. and Gossypium barbadense L. and includes all plant varietiesthat can be bred with cotton plants containing event MON 88702,including wild cotton species as well as those plants belonging to thegenus Gossypium that permit breeding between species.

The present invention provides for transgenic plants which have beentransformed with a DNA construct that contains an expression cassetteexpressing toxic amounts of insecticidal protein TIC8334_16. What ismeant by toxic amount is an efficacious amount, an insecticidal amount,an insecticidally effective amount, a target insect suppressive amount,an efficacious pesticidal amount, an amount in the diet of insects inthe order of Hemiptera or Thysanoptera that is insecticidal, and othersimilar terms to be understood according to conventional usage by thoseof ordinary skill in the relevant art. Cotton plants transformedaccording to the methods and with the DNA constructs disclosed hereinare resistant to Hemipteran and Thysanopteran insect pests.

A transgenic “plant” is produced by transformation of a plant cell withheterologous DNA, i.e., a polynucleic acid construct that includes anumber of efficacious features of interest; regeneration of a plantresulting from the insertion of the transgene into the genome of theplant cell, and selection of a particular plant characterized byinsertion into a particular genome location and the number ofefficacious features of the regenerated transgenic plant. The term“event” refers to the unique molecular sequence resulting from insertingtransgenic DNA into the gonome of a cotton plant at a specific location.This sequence is present in the original transformant, and comprises theinserted DNA, and flanking genomic sequence immediately adjacent to theinserted DNA. Such sequence is unique and would be expected to betransferred to a progeny that receives the inserted DNA including thetransgene of interest as the result of a sexual cross of parental linethat includes the inserted DNA (e.g., the original transformant andprogeny resulting from selfing) and a parental line that does notcontain the inserted DNA. The present invention also provides theoriginal transformant plant and progeny of the transformant that includethe heterologous DNA. Such progeny may be produced by a sexual outcrossbetween plants comprising the event and another plant wherein theprogeny includes the heterologous DNA. Even after repeated back-crossingto a recurrent parent, the event is present in the progeny of the crossat the same chromosomal location. The present invention is related tothe transgenic event, cotton plants comprising MON 88702, progenythereof, and DNA compositions contained therein.

Reference in this application to an “isolated DNA molecule”, or anequivalent term or phrase, is intended to mean that the DNA molecule isone that is present alone or in combination with other compositions, butnot within its natural environment. For example, nucleic acid elementssuch as a coding sequence, intron sequence, untranslated leadersequence, promoter sequence, transcriptional termination sequence, andthe like, that are naturally found within the DNA of the genome of anorganism are not considered to be “isolated” so long as the element iswithin the genome of the organism and at the location within the genomein which it is naturally found. However, each of these elements, andsubparts of these elements, would be “isolated” within the scope of thisdisclosure so long as the element is not within the genome of theorganism and at the location within the genome in which it is naturallyfound. Similarly, a nucleotide sequence encoding an insecticidal proteinor any naturally occurring insecticidal variant of that protein would bean isolated nucleotide sequence so long as the nucleotide sequence wasnot within the DNA of the bacterium from which the sequence encoding theprotein is naturally found. A synthetic nucleotide sequence encoding theamino acid sequence of the naturally occurring insecticidal proteinwould be considered to be isolated for the purposes of this disclosure.For the purposes of this disclosure, any transgenic nucleotide sequence,i.e., the nucleotide sequence of the DNA inserted into the genome of thecells of a plant or bacterium, or present in an extrachromosomal vector,would be considered to be an isolated nucleotide sequence whether it ispresent within the plasmid or similar structure used to transform thecells, within the genome of the plant or bacterium, or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the plant or bacterium.

A “probe” is an nucleic acid to which may be attached a conventionaldetectable label or reporter molecule, e.g., a radioactive isotope,ligand, chemiluminescent agent, or enzyme. Such a probe is complementaryto a strand of a target nucleic acid, in the case of the presentinvention, to a strand of DNA from MON 88702 whether from a MON 88702containing plant or from a sample that includes MON 88702 DNA. Probesaccording to the present invention include not only deoxyribonucleic orribonucleic acids, but also polyamides and other probe materials thatbind specifically to a target DNA sequence and can be used to detect thepresence of that target DNA sequence.

DNA primers are polynucleic acids that are annealed to a complementarytarget DNA strand by nucleic acid hybridization to form a hybrid betweenthe primer and the target DNA strand, then extended along the target DNAstrand by a polymerase, e.g., a DNA polymerase. A DNA primer pair or aDNA primer set of the present invention refer to two DNA primers usefulfor amplification of a target nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other conventional polynucleic acidamplification methods.

DNA probes and DNA primers are generally eleven (11) polynucleotides ormore in length, often eighteen (18) polynucleotides or more, twenty-four(24) polynucleotides or more, or thirty (30) polynucleotides or more.Such probes and primers are selected to be of sufficient length tohybridize specifically to a target sequence under high stringencyhybridization conditions. Preferably, probes and primers according tothe present invention have complete sequence similarity with the targetsequence, although probes differing from the target sequence that retainthe ability to hybridize to target sequences may be designed byconventional methods.

Primers and probes based on the flanking genomic DNA and insertsequences disclosed herein can be used to confirm (and, if necessary, tocorrect) the disclosed DNA sequences by conventional methods, e.g., byre-cloning and sequencing such DNA molecules.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA molecule. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic plant in a sample.Polynucleic acid molecules also referred to as nucleic acid segments orfragments thereof are capable of specifically hybridizing to othernucleic acid molecules under certain circumstances. As used herein, twopolynucleic acid molecules are said to be capable of specificallyhybridizing to one another if the two molecules are capable of formingan anti-parallel, double-stranded nucleic acid structure. A nucleic acidmolecule is said to be the “complement” of another nucleic acid moleculeif they exhibit complete complementarity. As used herein, molecules aresaid to exhibit “complete complementarity” when every nucleotide of oneof the molecules is complementary to a nucleotide of the other. Twomolecules are said to be “minimally complementary” if they can hybridizeto one another with sufficient stability to permit them to remainannealed to one another under at least conventional “low-stringency”conditions. Similarly, the molecules are said to be “complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under conventional“high-stringency” conditions. Conventional stringency conditions aredescribed by Sambrook et al., 1989, and by Haymes et al., In: NucleicAcid Hybridization, A Practical Approach, IRL Press, Washington, D.C.(1985). Departures from complete complementarity are thereforepermissible, as long as such departures do not completely preclude thecapacity of the molecules to form a double-stranded structure. In orderfor a nucleic acid molecule to serve as a primer or probe it need onlybe sufficiently complementary in sequence to be able to form a stabledouble-stranded structure under the particular solvent and saltconcentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions that promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In a preferredembodiment, a polynucleic acid of the present invention willspecifically hybridize to one or more of the nucleic acid molecules setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26,or complements thereof or fragments of either under moderately stringentconditions, for example at about 2.0×SSC and about 65° C. In aparticularly preferred embodiment, a nucleic acid of the presentinvention will specifically hybridize to one or more of the nucleic acidmolecules set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, orSEQ ID NO:26, or complements or fragments of either under highstringency conditions. In one aspect of the present invention, apreferred marker nucleic acid molecule of the present invention has thenucleic acid sequence set forth in SEQ ID NO:1, or SEQ ID NO:2, or SEQID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6, or SEQ ID NO:7,or SEQ ID NO:8, or SEQ ID NO:9, or SEQ ID NO:10, or SEQ ID NO:22, or SEQID NO:23, or SEQ ID NO:24, or SEQ ID NO:25, or SEQ ID NO:26 orcomplements thereof or fragments of either. The hybridization of theprobe to the target DNA molecule can be detected by any number ofmethods known to those skilled in the art, these can include, but arenot limited to, fluorescent tags, radioactive tags, antibody based tags,and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofpolynucleic acid amplification method directed to a target polynucleicacid molecule that is part of a polynucleic acid template. For example,to determine whether a cotton plant resulting from a sexual crosscontains transgenic plant genomic DNA from a cotton plant comprisingevent MON 88702 of the present invention, DNA that is extracted from acotton plant tissue sample may be subjected to a polynucleic acidamplification method using a primer pair that includes a first primerderived from a genomic DNA sequence in the region flanking theheterologous inserted DNA of event MON 88702 and is elongated bypolymerase 5′ to 3′ in the direction of the inserted DNA. The secondprimer is derived from the heterologous inserted DNA molecule iselongated by the polymerase 5′ to 3′ in the direction of the flankinggenomic DNA from which the first primer is derived. The amplicon mayrange in length from the combined length of the primer pair plus onenucleotide base pair, or plus about fifty nucleotide base pairs, or plusabout two hundred-fifty nucleotide base pairs, or plus about fourhundred-fifty nucleotide base pairs or more. Alternatively, a primerpair can be derived from genomic sequence on both sides of the insertedheterologous DNA so as to produce an amplicon that includes the entireinsert polynucleotide sequence (e.g., a forward primer isolated from thegenomic portion on the 5′ end of SEQ ID NO:10 and a reverse primerisolated from the genomic portion on the 3′ end of SEQ ID NO:10 thatamplifies a DNA molecule comprising the inserted DNA sequence (SEQ IDNO:9) identified herein in the event MON 88702 genome). A member of aprimer pair derived from the plant genomic sequence adjacent to theinserted transgenic DNA is located a distance from the inserted DNAsequence, this distance can range from one nucleotide base pair up toabout twenty thousand nucleotide base pairs. The use of the term“amplicon” specifically excludes primer dimers that may be formed in theDNA thermal amplification reaction.

For practical purposes, one should design primers which produceamplicons of a limited size range, for example, between 20 to 1000bases. Smaller (shorter polynucleotide length) sized amplicons ingeneral are more reliably produced in thermal amplification reactions,allow for shorter cycle times, and can be easily separated andvisualized on agarose gels or adapted for use in endpoint TAQMAN®-likeassays. Smaller amplicons can be produced and detected by methods knownin the art of DNA amplicon detection. In addition, amplicons producedusing the primer pairs can be cloned into vectors, propagated, isolated,and sequenced or can be sequenced directly with methods well establishedin the art. Any primer pair derived from the combination of SEQ ID NO:7and SEQ ID NO:9 or the combination of SEQ ID NO:8 and SEQ ID NO:9 thatare useful in a DNA amplification method to produce an amplicondiagnostic for MON 88702 or progeny thereof is an aspect of theinvention. Any single isolated DNA polynucleotide primer moleculecomprising at least 15 contiguous nucleotides of SEQ ID NO:7, or itscomplement that is useful in a DNA amplification method to produce anamplicon diagnostic for MON 88702 or progeny thereof is an aspect of theinvention. Any single isolated DNA polynucleotide primer moleculecomprising at least 15 contiguous nucleotides of SEQ ID NO:8, or itscomplement that is useful in a DNA amplification method to produce anamplicon diagnostic for plants comprising MON 88702 or progeny thereofis an aspect of the invention. Any single isolated DNA polynucleotideprimer molecule comprising at least 15 contiguous nucleotides of SEQ IDNO:9, or its complement that is useful in a DNA amplification method toproduce an amplicon diagnostic for MON 88702 or progeny thereof is anaspect of the invention.

Polynucleic acid amplification can be accomplished by any of the variouspolynucleic acid amplification methods known in the art, including thepolymerase chain reaction (PCR). Amplification methods are known in theart and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed.Innis et al., Academic Press, San Diego, 1990. PCR amplification methodshave been developed to amplify up to 22 kb (kilobase) of genomic DNA andup to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci.USA 91:5695-5699, 1994). These methods as well as other methods known inthe art of DNA amplification may be used in the practice of the presentinvention. The sequence of the heterologous DNA insert or flankinggenomic DNA sequence from cotton event MON 88702 can be verified (andcorrected if necessary) by amplifying such DNA molecules from cottonseed containing event MON 88702 DNA or cotton plants grown from thecotton seed containing event MON 88702 DNA deposited with the ATCChaving accession No. PTA-122520, using primers derived from thesequences provided herein, followed by standard DNA sequencing of thePCR amplicon or cloned DNA fragments thereof.

The diagnostic amplicon produced by these methods may be detected by aplurality of techniques. One such method is Genetic Bit Analysis(Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) where a DNAoligonucleotide is designed that overlaps both the adjacent flankinggenomic DNA sequence and the inserted DNA sequence. The oligonucleotideis immobilized in wells of a microtiter plate. Following PCR of theregion of interest (using one primer in the inserted sequence and one inthe adjacent flanking genomic sequence), a single-stranded PCR productcan be hybridized to the immobilized oligonucleotide and serve as atemplate for a single base extension reaction using a DNA polymerase andlabeled dideoxynucleotide triphosphates (ddNTPs) specific for theexpected next base. Readout may be fluorescent or ELISA-based. A signalindicates presence of the transgene/genomic sequence due to successfulamplification, hybridization, and single base extension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method, anoligonucleotide is designed that overlaps the adjacent genomic DNA andinsert DNA junction. The oligonucleotide is hybridized tosingle-stranded PCR product from the region of interest (one primer inthe inserted sequence and one in the flanking genomic sequence) andincubated in the presence of a DNA polymerase, ATP, sulfurylase,luciferase, apyrase, adenosine 5′ phosphosulfate and luciferin. DNTPsare added individually and the incorporation results in a light signalthat is measured. A light signal indicates the presence of thetransgene/genomic sequence due to successful amplification,hybridization, and single or multi-base extension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedthat overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene/genomic sequence due tosuccessful amplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed that overlaps thegenomic flanking and insert DNA junction. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermalstable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of thetransgene/genomic sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996). Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermalstablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization.

DNA detection kits that are based on DNA amplification methods containDNA primer molecules that hybridize specifically to a target DNA andamplify a diagnostic amplicon under the appropriate reaction conditions.The kit may provide an agarose gel based detection method or any numberof methods of detecting the diagnostic amplicon that are known in theart. DNA detection kits can be developed using the compositionsdisclosed herein and are useful for identification of cotton event MON88702 DNA in a sample and can be applied to methods for breeding cottonplants containing event MON 88702 DNA. A kit that contains DNA primersthat are homologous or complementary to any portion of the cottongenomic region as set forth in SEQ ID NO:10 and to any portion of theinserted transgenic DNA as set forth in SEQ ID NO:10 is an object of theinvention. The DNA molecules can be used in DNA amplification methods(PCR) or as probes in polynucleic acid hybridization methods, i.e.,southern analysis, northern analysis.

Junction sequences may be represented by a sequence from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, and SEQ ID NO:25. For example, the junctionsequences may be arbitrarily represented by the nucleotide sequencesprovided as SEQ ID NO:1 and SEQ ID NO:2. Alternatively, the junctionsequences may be arbitrarily represented by the nucleotide sequencesprovided as SEQ ID NO:3 and SEQ ID NO:4. Alternatively, the junctionsequences may be arbitrarily represented by the nucleotide sequencesprovided as SEQ ID NO:5 and SEQ ID NO:6. These nucleotides are connectedby phosphodiester linkage and in cotton event MON 88702 are present aspart of the recombinant plant cell genome. The identification of one ormore of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQID NO:24, and SEQ ID NO:25 in a sample derived from a cotton plant,cotton seed, or cotton plant part is determinative that the DNA wasobtained from cotton event MON 88702 and is diagnostic for the presencein a sample containing DNA from cotton event MON 88702. The inventionthus provides a DNA molecule that contains at least one of thenucleotide sequences provided as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. Anysegment of DNA derived from transgenic cotton event MON 88702 that issufficient to include at least one of the sequences provided as SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQID NO:25, and SEQ ID NO:26 is within the scope of the invention. Inaddition, any polynucleotide comprising a sequence complementary to anyof the sequences described within this paragraph is within the scope ofthe invention.

The invention provides exemplary DNA molecules that can be used eitheras primers or probes for detecting the presence of DNA derived from acotton plant comprising event MON 88702 DNA in a sample. Such primers orprobes are specific for a target nucleic acid sequence and as such areuseful for the identification of cotton event MON 88702 nucleic acidsequence by the methods of the invention described herein.

A “primer” is typically a highly purified, isolated polynucleotide thatis designed for use in specific annealing or hybridization methods thatinvolve thermal amplification. A pair of primers may be used withtemplate DNA, such as a sample of cotton genomic DNA, in a thermalamplification, such as polymerase chain reaction (PCR), to produce anamplicon, where the amplicon produced from such reaction would have aDNA sequence corresponding to sequence of the template DNA locatedbetween the two sites where the primers hybridized to the template. Asused herein, an “amplicon” is a replication of a piece or fragment ofDNA that has been synthesized using amplification techniques. In someembodiments, an amplicon of the invention may comprise at least one ofthe sequences provided as SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:17, SEQID NO:19. In another emodiment, an amplicon may comprise at least one ofthe junction sequences of the present event, such as SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,and SEQ ID NO:26. A primer is typically designed to hybridize to acomplementary target DNA strand to form a hybrid between the primer andthe target DNA strand, and the presence of the primer is a point ofrecognition by a polymerase to begin extension of the primer (i.e.,polymerization of additional nucleotides into a lengthening nucleotidemolecule) using as a template the target DNA strand. Primer pairs, asused in the invention, are intended to refer to use of two primersbinding opposite strands of a double stranded nucleotide segment for thepurpose of amplifying linearly the polynucleotide segment between thepositions targeted for binding by the individual members of the primerpair, typically in a thermal amplification reaction or otherconventional nucleic-acid amplification methods. Exemplary DNA moleculesuseful as primers are provided as SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:17, SEQ ID NO:19. The primer pair provided as SEQ ID NO:11 and SEQ IDNO:12 are useful as a first DNA molecule and a second DNA molecule thatis different from the first DNA molecule, and both are each ofsufficient length of contiguous nucleotides of SEQ ID NO:10 to functionas DNA primers that, when used together in a thermal amplificationreaction with template DNA derived from cotton event MON 88702, toproduce an amplicon diagnostic for cotton event MON 88702 DNA in asample.

A “probe” is a nucleic acid that is complementary to a strand of atarget nucleic acid. Probes according to the invention include not onlydeoxyribonucleic or ribonucleic acids but also polyamides and otherprobe materials that bind specifically to a target DNA sequence and thedetection of such binding can be useful in diagnosing, discriminating,determining, or confirming the presence of that target DNA sequence in aparticular sample. A probe may be attached to a conventional detectablelabel or reporter molecule, e.g., a radioactive isotope, ligand,chemiluminescent agent, or enzyme. Exemplary DNA molecules useful as aprobes are provided as SEQ ID NO:13 and SEQ ID NO:18.

Probes and primers according to the invention may have complete sequenceidentity with the target sequence, although primers and probes differingfrom the target sequence that retain the ability to hybridizepreferentially to target sequences may be designed by conventionalmethods. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed. Any conventional nucleic acidhybridization or amplification method can be used to identify thepresence of transgenic DNA from cotton event MON 88702 in a sample.Probes and primers are generally at least about 11 nucleotides, at leastabout 18 nucleotides, at least about 24 nucleotides, or at least about30 nucleotides or more in length. Such probes and primers hybridizespecifically to a target DNA sequence under stringent hybridizationconditions. Conventional stringency conditions are described by Sambrooket al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985).

Any number of methods well known to those skilled in the art can be usedto isolate and manipulate a DNA molecule, or fragment thereof, disclosedin the invention, including thermal amplification methods. DNAmolecules, or fragments thereof, can also be obtained by othertechniques such as by directly synthesizing the fragment by chemicalmeans, as is commonly practiced by using an automated oligonucleotidesynthesizer.

The DNA molecules and corresponding nucleotide sequences provided hereinare therefore useful for, among other things, identifying cotton eventMON 88702, selecting plant varieties or hybrids comprising cotton eventMON 88702, detecting the presence of DNA derived from the transgeniccotton event MON 88702 in a sample, and monitoring samples for thepresence and/or absence of cotton event MON 88702 or plant parts derivedfrom cotton plants comprising event MON 88702.

The invention provides cotton plants, cotton plant cells, cotton seeds,cotton plant parts (such as pollen, ovule, pod, flower tissue, roottissue, stem tissue, and leaf tissue), cotton progeny plants, cottonlint, and cotton commodity products. These cotton plants, cotton plantcells, cotton seeds, cotton plant parts, cotton progeny plants, cottonlint, and cotton commodity products contain a detectable amount of apolynucleotide of the invention, i.e., such as a polynucleotide havingat least one of the sequences provided as SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ IDNO:26. Cotton plants, plant cells, seeds, plant parts, and progenyplants of the invention may also contain one or more additionaltransgenes. Such additional transgene may be any nucleotide sequenceencoding a protein or RNA molecule conferring a desirable traitincluding but not limited to increased insect resistance, increasedwater use efficiency, increased yield performance, increased droughtresistance, increased seed quality, and/or increased herbicidetolerance, in which the desirable trait is measured with respect to acotton plant lacking such additional transgene.

The invention provides cotton plants, cotton plant cells, cotton seeds,cotton plant parts (such as pollen, ovule, pod, flower tissue, roottissue, stem tissue, and leaf tissue), cotton progeny plants derivedfrom a transgenic cotton plant containing event MON 88702 DNA. Arepresentative sample of cotton seed containing event MON 88702 DNA hasbeen deposited according to the Budapest Treaty with the American TypeCulture Collection (ATCC®). The ATCC repository has assigned the PatentDeposit Designation PTA-122520 to the seed containing event MON 88702DNA.

The invention provides a microorganism comprising a DNA molecule havingat least one sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26present in its genome. An example of such a microorganism is atransgenic plant cell. Microorganisms, such as a plant cell of theinvention, are useful in many industrial applications, including but notlimited to: (i) use as research tool for scientific inquiry orindustrial research; (ii) use in culture for producing endogenous orrecombinant carbohydrate, lipid, nucleic acid, or protein products orsmall molecules that may be used for subsequent scientific research oras industrial products; and (iii) use with modern plant tissue culturetechniques to produce transgenic plants or plant tissue cultures thatmay then be used for agricultural research or production. The productionand use of microorganisms such as transgenic plant cells utilizes modernmicrobiological techniques and human intervention to produce a man-made,unique microorganism. In this process, recombinant DNA is inserted intoa plant cell's genome to create a transgenic plant cell that is separateand unique from naturally occurring plant cells. This transgenic plantcell can then be cultured much like bacteria and yeast cells usingmodern microbiology techniques and may exist in an undifferentiated,unicellular state. The transgenic plant cell's new genetic compositionand phenotype is a technical effect created by the integration of theheterologous DNA into the genome of the cell. Another aspect of theinvention is a method of using a microorganism of the invention. Methodsof using microorganisms of the invention, such as transgenic plantcells, include (i) methods of producing transgenic cells by integratingrecombinant DNA into the genome of the cell and then using this cell toderive additional cells possessing the same heterologous DNA; (ii)methods of culturing cells that contain recombinant DNA using modernmicrobiology techniques; (iii) methods of producing and purifyingendogenous or recombinant carbohydrate, lipid, nucleic acid, or proteinproducts from cultured cells; and (iv) methods of using modern planttissue culture techniques with transgenic plant cells to producetransgenic plants or transgenic plant tissue cultures.

Plants of the invention may pass along the event MON 88702 DNA,including the transgene inserted in cotton event MON 88702, to progeny.As used herein, “progeny” includes any plant, plant cell, seed, and/orregenerable plant part containing the event MON 88702 DNA derived froman ancestor plant and/or comprising a DNA molecule having at least onesequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. Plants,progeny, and seeds may be homozygous or heterozygous for the transgeneof event MON 88702. Progeny may be grown from seeds produced by a cottonevent MON 88702 containing plant and/or from seeds produced by a plantfertilized with pollen from a cotton event MON 88702 containing plant.

Progeny plants may be self-pollinated (also known as “selfing”) togenerate a true breeding line of plants, i.e., plants homozygous for thetransgene. Selfing of appropriate progeny can produce plants that arehomozygous for both added, exogenous genes.

Alternatively, progeny plants may be out-crossed, e.g., bred withanother unrelated plant, to produce a varietal or a hybrid seed orplant. The other unrelated plant may be transgenic or non-transgenic. Avarietal or hybrid seed or plant of the invention may thus be derived bysexually crossing a first parent that lacks the specific and unique DNAof the cotton event MON 88702 with a second parent comprising cottonevent MON 88702, resulting in a hybrid comprising the specific andunique DNA of the cotton event MON 88702. Each parent can be a hybrid oran inbred/varietal, so long as the cross or breeding results in a plantor seed of the invention, i.e., a seed having at least one allelecontaining the DNA of cotton event MON 88702 and/or a DNA moleculehaving at least one sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQID NO:26. Two different transgenic plants may thus be crossed to producehybrid offspring that contain two independently segregating, added,exogenous genes. For example, the MON 88702 containing TIC834_16conferring insect resistance to cotton can be crossed with othertransgenic cotton plants to produce a plant having the characteristicsof both transgenic parents. One example of this would be a cross of MON88702 containing TIC834_16 conferring Hemipteran and Thysanopteraninsect resistance to cotton with a plant having one or more additionaltraits such as herbicide tolerance (e.g., cotton event MON 88913(MON-88913-8) or cotton event LLCotton25 (ACS-GHØØ1-3)) and/or insectcontrol (e.g., cotton event MON 15985 (MON-15985-7)), resulting in aprogeny plant or seed that has resistance to Hemipteran andThysanopteran insect pests and has at least one or more additionaltraits. Back-crossing to a parental plant and out-crossing with anon-transgenic plant are also contemplated, as is vegetativepropagation. Descriptions of other breeding methods that are commonlyused for different traits and crops can be found in one of severalreferences, e.g., Fehr, in Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

The invention provides a plant part that is derived from cotton plantscomprising event MON 88702. As used herein, a “plant part” refers to anypart of a plant which is comprised of material derived from a cottonplant comprising event MON 88702. Plant parts include but are notlimited to pollen, ovule, pod, flower, root or stem tissue, fibers, andleaves. Plant parts may be viable, nonviable, regenerable, and/ornonregenerable.

The invention provides a commodity product that is derived from cottonplants comprising event MON 88702 and that contains a detectable amountof a nucleic acid specific for event MON 88702. As used herein, a“commodity product” refers to any composition or product which iscomprised of material derived from a cotton plant, whole or processedcotton seed, one or more plant cells and/or plant parts containing thecotton event MON 88702 DNA. Commodity products may be sold to consumersand may be viable or nonviable. Nonviable commodity products include butare not limited to nonviable seeds; whole or processed seeds, seedparts, and plant parts; cotton fiber, cotton lint, cotton oil andderivatives of cotton oil such as shortening, soaps, and cosmetics,cotton protein, cotton meal, animal feed comprising cotton, papercomprising cotton, cotton biomass, candle wicks, string and rope, cottonballs, cotton batting, cellulose products such as rayon, plastics,photographic film, cellophane, fatty acids used for various industrialuses such as insulation materials, linoleum, oilcloth, waterproofing,and as a paint base, and fuel products produced using cotton plants andcotton plant parts. Viable commodity products include but are notlimited to seeds, plants, and plant cells. The cotton plants comprisingevent MON 88702 can thus be used to manufacture any commodity producttypically acquired from cotton. Any such commodity product that isderived from cotton plants comprising event MON 88702 may contain atleast a detectable amount of the specific and unique DNA correspondingto cotton event MON 88702, and specifically may contain a detectableamount of a polynucleotide comprising a DNA molecule having at least onesequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. Any standardmethod of detection for nucleotide molecules may be used, includingmethods of detection disclosed herein. A commodity product is within thescope of the invention if there is any detectable amount of a DNAmolecule having at least one sequence selected from SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,and SEQ ID NO:26 in the commodity product.

The plants, progeny, seeds, plant cells, plant parts (such as pollen,ovule, pod, flower, root or stem tissue, and leaves), and commodityproducts of the invention are therefore useful for, among other things,growing plants for the purpose of producing seed and/or plant partscomprising cotton event MON 88702 for agricultural purposes, producingprogeny comprising cotton event MON 88702 for plant breeding andresearch purposes, use with microbiological techniques for industrialand research applications, and sale to consumers.

Methods for producing an insect resistant cotton plant comprising theDNA sequences specific and unique to event MON 88702 of the inventionare provided. Transgenic plants used in these methods may be homozygousor heterozygous for the transgene. Progeny plants produced by thesemethods may be varietal or hybrid plants; may be grown from seedsproduced by a cotton event MON 88702 containing plant and/or from seedsproduced by a plant fertilized with pollen from a cotton event MON 88702containing plant; and may be homozygous or heterozygous for thetransgene. Progeny plants may be subsequently self-pollinated togenerate a true breeding line of plants, i.e., plants homozygous for thetransgene, or alternatively may be out-crossed, e.g., bred with anotherunrelated plant, to produce a varietal or a hybrid seed or plant.

Methods of detecting the presence of DNA derived from a cotton cell,cotton tissue, cotton seed, or cotton plant comprising cotton event MON88702 in a sample are provided. One method consists of (i) extracting aDNA sample from at least one cotton cell, cotton tissue, cotton seed, orcotton plant, (ii) contacting the DNA sample with at least one primerthat is capable of producing DNA sequence specific to event MON 88702DNA under conditions appropriate for DNA sequencing, (iii) performing aDNA sequencing reaction, and then (iv) confirming that the nucleotidesequence comprises a nucleotide sequence specific for event MON 88702,or the construct comprised therein, such as one selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. Another methodconsists of (i) extracting a DNA sample from at least one cotton cell,cotton tissue, cotton seed, or cotton plant, (ii) contacting the DNAsample with a primer pair that is capable of producing an amplicon fromevent MON 88702 DNA under conditions appropriate for DNA amplification,(iii) performing a DNA amplification reaction, and then (iv) detectingthe amplicon molecule and/or confirming that the nucleotide sequence ofthe amplicon comprises a nucleotide sequence specific for event MON88702, such as one selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. Theamplicon should be one that is specific for event MON 88702, such as anamplicon that comprises SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, orSEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6. The detection of anucleotide sequence specific for event MON 88702 in the amplicon isdeterminative and/or diagnostic for the presence of the cotton event MON88702 specific DNA in the sample. An example of a primer pair that iscapable of producing an amplicon from event MON 88702 DNA underconditions appropriate for DNA amplification is provided as SEQ IDNO:11, and SEQ ID NO:12. Other primer pairs may be readily designed byone of skill in the art and would produce an amplicon comprising SEQ IDNO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, orSEQ ID NO:6, wherein such a primer pair comprises at least one primerwithin the genomic region flanking the insert and a second primer withinthe insert. Another method of detecting the presence of DNA derived froma cotton cell, cotton tissue, cotton seed, or cotton plant comprisingcotton event MON 88702 in a sample consists of (i) extracting a DNAsample from at least one cotton cell, cotton tissue, cotton seed, orcotton plant, (ii) contacting the DNA sample with a DNA probe specificfor event MON 88702 DNA, (iii) allowing the probe and the DNA sample tohybridize under stringent hybridization conditions, and then (iv)detecting hybridization between the probe and the target DNA sample. Anexample of the sequence of a DNA probe that is specific for event MON88702 DNA is provided as SEQ ID NO:13 or SEQ ID NO:18. Other probes maybe readily designed by one of skill in the art and would comprise atleast one fragment of genomic DNA flanking the insert and at least onefragment of insert DNA, such as sequences provided in, but not limitedto, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, and SEQ ID NO:10. Detection of probe hybridization to the DNAsample is diagnostic for the presence of cotton event MON 88702 specificDNA in the sample. Absence of hybridization is alternatively diagnosticof the absence of cotton event MON 88702 specific DNA in the sample.

DNA detection kits are provided that are useful for the identificationof cotton event MON 88702 DNA in a sample and can also be applied tomethods for breeding cotton plants containing the appropriate event DNA.Such kits contain DNA primers and/or probes comprising fragments of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, and SEQ ID NO:26. One example of such a kitcomprises at least one DNA molecule of sufficient length of contiguousnucleotides of SEQ ID NO:10 to function as a DNA probe useful fordetecting the presence and/or absence of DNA derived from transgeniccotton plants comprising event MON 88702 in a sample. The DNA derivedfrom transgenic cotton plants comprising event MON 88702 would comprisea DNA molecule having at least one sequence selected from SEQ ID NO:1,SEQ ID NO 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26. A DNA molecule sufficient for use as a DNAprobe is provided that is useful for determining, detecting, ordiagnosing the presence and/or absence of cotton event MON 88702 DNA ina sample is provided as SEQ ID NO:13. Other probes may be readilydesigned by one of skill in the art and should comprise at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, or at least 40 contiguous nucleotides of SEQ IDNO:10 and be sufficiently unique to cotton event MON 88702 DNA in orderto identify DNA derived from the event. Another type of kit comprises aprimer pair useful for producing an amplicon useful for detecting thepresence and/or absence of DNA derived from transgenic cotton event MON88702 in a sample. Such a kit would employ a method comprisingcontacting a target DNA sample with a primer pair as described herein,then performing a nucleic acid amplification reaction sufficient toproduce an amplicon comprising a DNA molecule having at least onesequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22,SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, and thendetecting the presence and/or absence of the amplicon. Such a method mayalso include sequencing the amplicon or a fragment thereof, which wouldbe determinative of, i.e. diagnostic for, the presence of the cottonevent MON 88702 specific DNA in the target DNA sample. Other primerpairs may be readily designed by one of skill in the art and shouldcomprise at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25, at least 26, at least 27, at least 28, at least 29, or atleast 30 contiguous nucleotides of sequences provided in, but notlimited to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26 and be sufficientlyunique to cotton event MON 88702 DNA in order to identify DNA derivedfrom the event.

The kits and detection methods of the invention are useful for, amongother things, identifying cotton event MON 88702, selecting plantvarieties or hybrids comprising cotton event MON 88702, detecting thepresence of DNA derived from the transgenic cotton plants comprisingevent MON 88702 in a sample, and monitoring samples for the presenceand/or absence of cotton plants comprising event MON 88702 or plantparts derived from cotton plants comprising event MON 88702.

The sequence of the heterologous DNA insert, junction sequences, orflanking sequences from cotton event MON 88702 can be verified (andcorrected if necessary) by amplifying such sequences from the eventusing primers derived from the sequences provided herein followed bystandard DNA sequencing of the amplicon or of the cloned DNA.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Deposit Information

A deposit of a representative sample of cotton seed containing event MON88702 was made on Sep. 11, 2015 according to the Budapest Treaty withthe American Type Culture Collection (ATCC) having an address at 10801University Boulevard, Manassas, Va. USA, Zip Code 20110, and assignedATCC Accession No. PTA-122520. Access to the deposits will be availableduring the pendency of the application to the Commissioner of Patentsand Trademarks and persons determined by the Commissioner to be entitledthereto upon request. Upon issuance of the patent, all restrictions uponavailability to the public will be irrevocably removed. The deposit willbe maintained in the depository for a period of 30 years, or 5 yearsafter the last request, or for the effective life of the patent,whichever is longer, and will be replaced as necessary during thatperiod.

EXAMPLES Example 1 Selection and Molecular Characterization of CottonEvent MON 88702

This example describes the transformation and selection of cotton eventMON 88702. The expression of transgenes in plants is known to beinfluenced by chromosomal insertion position, perhaps due to chromatinstructure (e.g., heterochromatin) or the proximity of transcriptionalregulation elements (e.g., enhancers) close to the integration site(Kurt Weising et al., (1988) Foreign genes in plants: transfer,structure, expression and applications. Annu. Rev. Genet. 22: 421-77).For this reason, it is often necessary to screen a large number oftransformation events in order to identify an event which demonstratesoptimal expression of the introduced gene of interest. For example, ithas been observed in plants and in other organisms that there may bewide variation in the levels of expression of an introduced gene amongevents. There may also be differences in spatial or temporal patters ofexpression, for example, differences in the relative expression of atransgene in various plant tissues, that may not correspond to thepatterns expected from transcriptional regulatory elements present inthe introduced gene construct.

For these reasons, the development of a transgenic cotton plantcomprising a insecticidal protein that was active against Hemipteransand Thysanopterans without any negative effects on agronomics, yield orstacking viability required extensive analyses over several years ofapproximately two thousand four hundred (2,400) events derived fromthirty nine (39) different plasmid vector constructs. The individualexpression cassettes in the (39) thirty nine plasmid constructs variedwith respect to the coding sequences for the insecticidal toxin proteinby either utilizing the TIC834_16 coding sequence, the TIC807 (U.S. Pat.No. 8,609,936) coding sequence, or coding sequences of variantinsecticidal toxin proteins closely related to TIC834_16, referred to as“engineered Hemipteran toxin proteins” and referenced as variants ofTIC807 in United States Patent Application US20130269060. The individualexpression cassettes in the thirty nine (39) plasmid constructs alsovaried with respect to the use of different transcriptional regulationelements and whether or not the insecticidal toxin protein was targetedto the chloroplast.

The transgene cassette presented as SEQ ID NO:27 is the construct usedto produce cotton event MON 88702. Numerous rounds of testing andcomparison of various plasmid vector constructs revealed that thistransgenic cassette produced the events with the best efficacy againstthe two Lygus species Lygus hesperus (Western tarnished plant bug) andLygus lineolaris (Tarnished plant bug), when compared to the eventsproduced by other transgene cassettes from the thirty eight (38) otherconstructs.

The transgenic Hemipteran and Thysanopteran insect resistant cottonevent MON 88702 was created through an Agrobacterium-mediatedtransformation of cotton meristem tissue (U.S. Pat. No. 8,044,260) witha two T-DNA system. In this system, an Agrobacterium strain employingone binary plasmid vector with two T-DNA transgene cassettes wasutilized. The first T-DNA transgene cassette was used for the selectionof transformed cotton plant cells using a selectable marker whichconfers tolerance to the antibiotic spectinomycin. The second T-DNAtransgene cassette—which is presented as SEQ ID NO:27 and demonstratedin FIG. 1b —was inserted into the cotton genome to confer insecticidalresistance to Hemipteran and Thysanopteran insect pests.

The second T-DNA transgene cassette presented as SEQ ID NO:27 and shownin FIG. 1b is comprised of a right T-DNA border (RB), an enhancer (E)derived from the FMV 35S promoter, operably linked 5′ to a promoter (P)derived from the Arabidopsis thaliana heat shock protein 81.2 forming achimeric FMV/Hsp81.2 promoter (U.S. Pat. No. 8,940,962), which isoperably linked 5′ to a 5′ UTR (L) derived from the Arabidopsis thalianaheat shock protein 81.2, which is operably linked 5′ to the TIC834_16coding sequence (United States Patent Application, US20130269060), whichis operably linked 5′ to a 3′ UTR (T) derived from the 35S Cauliflowermosaic virus gene VI, followed by a left T-DNA border (LB). The firstT-DNA transgene selection cassette inserted randomly into the cottongenome and at a site separate from the site of integration of the secondT-DNA transgene cassette containing the TIC834_16 expression cassette,thus allowing for segregation of the two T-DNA segments within thegenome of the transformed cotton plants during the process of selfingand/or backcrossing, e.g. screening R₁ and higher generation transgenicplants.

To produce MON 88702, cotton variety DP393 (Deltapine, St. Louis, Mo.)was transformed using the two T-DNA binary system described above.Transformed cotton cells were selected using spectinomycin andregenerated into intact cotton plants. Rooted plants with normalphenotypic characteristics were selected and transferred to soil forgrowth and further assessment. Initially, two hundred fifteen (215)events were created from the two T-DNA transformation system with afirst T-DNA transgene selection cassette and a second T-DNA transgenecassette containing the TIC834_16 expression cassette of SEQ ID NO:27.

After preliminary molecular characterization, one hundred thirty three(133) events were identified as sufficient quality for furtherassessment and testing. The events were analyzed using a combination ofanalytical techniques such as TaqMan and/or PCR analysis to determinethe presence of the second T-DNA transgene cassette containing theTIC834_16 expression cassette and the cassette copy number. From thisanalysis, thirty five (35) single-copy, R₁ events were selected and weregrown in the growth chamber and assayed for efficacy against Lyguslineolaris (Tarnished plant bug). The assay was conducted in three (3)separate experiments due to the size limitation of the growth chambers.

The thirty five (35) events were further characterized in more detailboth molecularly and phenotypically. Plants demonstrating efficacy werethen allowed to self-pollinate and set seed for further characterizationand analysis of the R₂ and subsequent generations. From the furtherefficacy screening, twenty four (24) events were selected for furtheranalysis and characterization. Each event was analyzed by Southern blotto determine copy number of the transgene cassette containing theTIC834_16 expression cassette of SEQ ID NO:27, the insertion pointwithin the cotton genome and the presence of backbone. If the presenceof a backbone was detected, the event was further analyzed using DNAamplification techniques to determine if the backbone was linked to thetransgene cassette containing the TIC834_16 expression cassette of SEQID NO:27 and if one kilobase of sequence flanking the left and rightborders were in a native gene. From this analysis twelve (12) eventsremained.

Of the twelve (12) events, one event demonstrated insertion of the leftborder into a native gene (Event 12), four events demonstrated insertionof the right border into a native gene (Events 3, 7, 9, and 10), andfive events were unable to be characterized for flanking sequence(Events 5, 6, 8, and 9). All twelve (12) events were used in subsequentefficacy testing in order to provide comparators for performance.Specifically, the twelve (12) events were tested and analyzed for insectresistance efficacy in growth chamber whole plant cage studies, cottonyield from transformed cotton plants, and protein expression.

Insect Resistance Efficacy

To assay for efficacy against Lygus lineolaris (Tarnished plant bug),five R₁ seeds were shown in 10 inch pots for each of the twelve (12)transgenic cotton events. An untransformed DP393 cotton variety was usedas a negative control. Plants were maintained in an environment chamberwith a photoperiod of sixteen (16) hours of light at thirty two (32)degrees Celsius and eight (8) hours of dark at twenty three (23) degreesCelsius, and a light intensity between eight hundred (800) and ninehundred (900) micro-Einsteins. At forty (40) to forty five (45) daysafter planting, the individual plants were enclosed in a cage made frombreathable plastic “pollination” sheets (Vilutis and Company Inc,Frankfort, Ill.). The sheet sleeves were secured to the main stem justabove the soil surface using a Velcro® tie. Two pairs of sexually maturemale and female Lygus lineolaris adults (six days old) from a laboratoryculture were collected into a fourteen milliliter round-bottom plastictube (Bacton Dickson Labware, Franklin Lakes, N.J.) and used for eachplant. The adults were released into each individual cage through asmall slit on the cage side and then the cage was securely closedensuring the insects would not escape. The insects were allowed to mateand the plants were kept in the cage for twenty one (21) days.

After twenty one (21) days, the plants were then cut below the cages andmoved to a laboratory where the insects were collected for each plantand counted. Before opening the cage, the plants were vigorously shakento ensure all of the insects fell off from their feeding sites to thebase of the cage. Then the cage base was opened and all plant materialremoved and placed on a black sheet. The insects were collected using anaspirator. The plant was then thoroughly inspected to recover anyremaining insects. The number of insects collected and theirdevelopmental stage were recorded for each plant. The insect counts weredivided into several groups based upon maturity of the Lygus: nymphs upto 3^(rd) instar, 4^(th) instar, 5^(th) instar and adults. Table 1 showsthe average number of Lygus for each of the twelve events wherein “SEM”is the standard error of the mean.

TABLE 1 Average number of Lygus lineolaris (Tarnished plant bug)recovered from caged cotton plants transformed with the transgenecassette containing the TIC834_16 expression cassette of SEQ ID NO:27.Number of Nymphal Instars Total Plants ≤3 SEM 4th SEM 5th SEM Adults SEMTPB/Plant SEM Event 1 5 4.20 2.62 3.00 1.52 2.20 1.02 0.00 0.00 9.404.82 Event 2 5 1.00 0.77 0.00 0.00 0.80 0.37 0.20 0.20 2.00 1.30 MON88702 Event 3 5 6.60 4.60 2.80 1.07 1.40 0.51 0.00 0.00 10.80 5.15 Event4 5 6.60 4.89 3.20 1.59 0.00 0.00 0.40 0.24 10.20 5.40 Event 5 5 2.002.00 0.00 0.00 0.00 0.00 0.00 0.00 2.00 2.00 Event 6 5 3.00 0.95 2.400.60 1.60 0.51 0.00 0.00 7.00 1.58 Event 7 5 0.00 0.00 0.40 0.24 0.000.00 0.00 0.00 0.40 0.24 Event 8 5 1.00 0.77 0.00 0.00 0.00 0.00 0.000.00 1.00 0.77 Event 9 5 3.80 2.65 2.00 1.05 2.20 0.66 0.40 0.24 8.404.12 Event 10 5 1.60 1.12 0.60 0.60 0.60 0.60 0.00 0.00 2.80 1.71 Event11 5 1.80 1.36 2.00 1.30 2.00 1.14 0.20 0.20 6.00 3.82 Event 12 5 0.400.40 0.60 0.40 1.60 1.36 1.40 0.87 4.00 2.79 DP393 10 7.90 2.10 5.400.91 7.25 1.28 5.20 1.36 25.75 3.50

As demonstrated in Table 1, all of the events expressing TIC834_16demonstrated resistance to Lygus lineolaris. Event 2/MON 88702 providedsuperior control of Lygus in this assay.

Cotton Yield from Transformed Cotton Plants

To assess yield, the twelve (12) events were grown in the field to fullmaturity. Four (4) rows of cotton plants were grown for each event.Cotton was harvested at two different time points during the growthcycle for each row. Events were assessed for the presence of theTIC834_16. If a row had plants lacking TIC834_16, the entire row wasexcluded from the yield assay. The yield for each event is provided inTable 2 and is expressed as pounds of seed cotton (lb S.C.). Asdemonstrated in Table 2, cotton event MON 88702 provided superior yield.

TABLE 2 Yield from cotton plants transformed with the transgene cassettecontaining the TIC834_16 expression cassette of SEQ ID NO: 27. Nurseryyield (lb S.C.) Number 1st 2nd Event of Rows harvest harvest Total Event1 4 24.5 0.0 24.5 Event 2 3 18.1 9.0 27.1 MON 88702 Event 3 1 8.5 0.08.5 Event 4 4 19.5 6.5 26 Event 5 4 26.6 0.0 26.6 Event 6 4 22.8 6.429.2 Event 7 4 19.7 13.3 27.8 Event 8 3 18 6.0 24 Event 9 4 15.4 5.721.1 Event 10 4 20.4 9.1 29.5 Event 11 4 24.4 4.8 29.2 Event 12 4 29.80.0 29.8

Protein Expression

Protein expression was determined using plants derived from R₂ seed.Plants were grown in the greenhouse for each of the twelve (12) eventsbeing assessed. Tissues were sampled at two different time points. Leafand squares were harvested at forty five (45) days after planting (45Land 45S, respectively), and leaf was harvested at seventy (70) daysafter planting (70L). Protein expression was measured using ELISA onlyophilized tissue and was expressed as parts per million (ppm).

The average expression of each event is provided in Table 3. Asdemonstrated in Table 3, the expression of TIC834_16 varies with eachevent and within each tissue. The level of expression did notnecessarily correlate with the plants' level of resistance to lygus.

TABLE 3 Average protein expression in selected tissues of R₂ cottonplants transformed with the transgene cassette containing the TIC834_16expression cassette of SEQ ID NO: 27. Protein Expression (dry weightppm) Event 45 L 45 S 70 L Event 1 918 746 872 Event 2 965 994 765 MON88702 Event 3 1183 1096 789 Event 4 1269 1088 958 Event 5 900 689 748Event 6 798 958 631 Event 7 1588 1574 1118 Event 8 1748 1468 1142 Event9 688 1072 580 Event 10 1074 1030 791 Event 11 868 1195 938 Event 12 976769 748

After several years of extensive characterization, two events stood outfrom the rest as providing high resistance to lygus, beneficialphenotypic characteristics, and superior yield relative to untransformedDP393; Event 2 (MON 88702) and Event 10. The left and right flankingsequences of both events were used to BLAST against the proprietaryassemblies of both the upland cotton TM1 (Gossypium hirsutum) tetraploidgenome and the Cotton D (Gossypium raimondii) diploid genome to identifyany potential issues with insertion into identified cotton genes, thuspotentially disrupting a native gene.

Insertion of a transgenic cassette into a native gene can be problematicwith respect to approval by regulatory agencies. Such observations cancause a product to not be approved or for extensive experimentation andcharacterization be performed to demonstrate the insertion within thenative gene does not cause deleterious effects in the plant. Event 2/MON88702 did not appear to be inserted within a gene based upon the uplandcotton TM1 assembly and the Cotton D diploid genome. In contrast, whileEvent 10 did not appear to insert within a gene based upon the uplandcotton TM1 assembly, based upon the Cotton D assembly it appeared theinsertion of the transgenic cassette occurred within the second intronof an endogenous gene. Thus, Event 10 had a transgenic cassetteinsertion into an endogenous cotton gene.

Because Event 2/MON 88702 did not have an insertion issue, providedsuperior yield as compared to Event 10 in field studies, had preferredmolecular characterization, and demonstrated excellent performance overseveral years in cage and field trails (as presented above and in theExamples below), it was identified as the superior event and thush theevent selected for commercialization.

Example 2 Cotton Event MON 88702 Demonstrates Resistance to ArtificialInfestations of Lygus hesperus (Western Tarnished Plant Bug) and Lyguslineolaris (Tarnished Plant Bug) in Open Field Cage Trials

MON 88702 provides resistance to the Lygus species Lygus hesperus(Western tarnished plant bug) and Lygus lineolaris (Tarnished plant bug)as demonstrated in this Example. Open field caging experiments wereconducted during consecutive growing seasons. Trials against Lyguslineolaris were conducted in Illinois in year 1 and repeated inTennessee in year 2. Trials against Lygus hesperus were conducted inArizona in year 1 and repeated in Arizona in year 2. Seeds from cottonevent MON 88702 and the negative control DP393 treated with Acceleron®(which contains fungicides, the insecticide Imidacloprid, and thenematacide Thiodicarb) were sown in two (2) fifteen (15) foot row plots.At fifteen (15) to twenty (20) days after planting, plants were thinnedto achieve uniform stand. At thirty five (35) to forty (40) days afterplanting, individual plants were carefully examined to remove any pestsand predators present. Once all pests and predators were removed, theplants were enclosed in a cage (150×228 centimeters) made from whitesolid voile (JoAnne Fabrics, Item Number 8139875). The bottom voilesleeve was secured to the main plant stem just above the soil surfaceand the top sleeve was tied together using a Velcro® tie. Metal poleswere placed at the end of each row and the top of each cage was attachedto a string to keep the cages in an upright position.

Lygus nymphs were collected from alfalfa or canola fields and reared inthe laboratory. On the day of eclosion, the adults were collected andmoved to a new container to keep track of their developmental stage. Atleast one hundred (100) to one hundred fifty (150) adults at anapproximately 1:1 sex ratio were placed in big plastic containers thatprovided uniform cohort for successful mating. On the day ofinfestation, all adults were at least six (6) days old with the cottonplants being forty five (45) to fifty (50) days after planting (i.e.,the peak squaring stage). Two pairs of male and female adults werecollected into fifteen milliliter plastic tubes and brought to thefield.

Each cage was opened from the top and re-examined to remove any pestsand predators which were overlooked in the previous inspection or whichhatched after caging. The plants were then infested by releasing thelygus adults into the cage. Eight (8) plants were infested pertreatment. Insects were allowed to reproduce and the resulting progenyallowed to feed on the caged cotton plant for thirty (30) days.Following this time period, the plants were harvested and the presenceof lygus for each plant was collected and recorded as described inExample 1.

Table 4 and Table 5 show the average next generation insect counts foryears 1 and 2 for Lygus lineolaris and Lygus hesperus, respectfully. Theinsect counts were divided into several groups based upon maturity ofthe lygus: small nymphs (defined as nymphs up to 3^(rd) instar), largenymphs (defined as being at the 4^(th) instar or the 5^(th) instar) andadults. Tables 4 and 5 show the average number of lygus for MON 88702and the negative control DP393 for each trail, wherein “SEM” is thestandard error of the mean.

TABLE 4 Average next generation insect counts for cotton event MON 88702and the negative control DP393 infested with Lygus lineolaris. NumberTotal of Small Large Total Lygus Year Event Plants Nymphs Nymphs AdultsLygus SEM 1 MON 88702 8 1.50 0.25 0.25 2.00 0.71 DP393 8 12.00 2.50 0.3814.88 1.95 2 MON 88702 8 0.13 1.25 2.13 3.50 1.72 DP393 16 4.19 4.133.38 11.69 2.06

TABLE 5 Average next generation insect counts for cotton event MON 88702and the negative control DP393 infested with Lygus hesperus. NumberTotal of Small Large Total Lygus Year Event Plants Nymphs Nymphs AdultsLygus SEM 1 MON 88702 8 0.00 0.50 1.75 2.25 1.32 DP393 8 2.13 8.38 11.6322.13 4.96 2 MON 88702 8 0.88 1.38 2.75 5.00 1.88 DP393 16 3.19 8.697.94 19.81 2.86

As demonstrated in Tables 4 and 5, cotton transgenic event MON 88702provides protection against both Lygus lineolaris and Lygus hesperus.The average insect counts for small nymphs, large nymphs and adults weremuch lower in the MON 88702 caged plants than the control DP393 plants.

Example 3 Cotton Event MON 88702 Provides Resistance to Lygus lineolarisand Lygus hesperus in the Field Under Natural Lygus Pressure

This Example demonstrates the ability of transgenic cotton event MON88702 to provide resistance to the Hemipteran pest species Lygushesperus (Western tarnished plant bug) and Lygus lineolaris (Tarnishedplant bug).

Field trials were conducted over 3 years: year 1, year 2 and year 3 invarious locations which experienced different levels of natural Lyguspressure as defined by an economic threshold determined through two (2)methods of survey. For fields in the Delta region of the United States(Arkansas (AR), Louisiana (LA), Mississippi (MS), and Tennessee (TN)),the drop cloth method was used to survey for the predominant Lygusspecies, Lygus lineolaris (Tarnished plant bug). The drop cloth methodessentially consists of placing a drop cloth between two rows of cottonand vigorously shaking the plants from both rows over the drop cloth.The insects are separated by stage and counted. For fields in theSouthwest region of the United States (Arizona (AZ) and California(CA)), the sweep net method was employed and used to survey thepredominant Lygus species, Lygus hesperus (Western tarnished plant bug).The sweep net survey method essentially consists of sweeping a netthrough the top of the canopy for a specified number of sweeps and thencounting the number of insects after separating for stages.

Eight (8) row plots of cotton were grown using 120 seeds treated withAcceleron® (which contained fungicides, the insecticide Imidacloprid,and the nematacide Thiodicarb) per thirty (30) foot long row for eachevent tested. Each plot was replicated four (4) times in the field. Theuntransformed cotton variety DP393 was used as a negative control andplanted in four (4) replicates of eight (8) row plots just as thetransgenic events. The Lygus nymphs sampled were divided into twogroups, small nymphs (defined as nymphs up to 3^(rd) instar) and largenymphs (defined as being at the 4^(th) instar or the 5^(th) instar). Thenumber of nymphs detected was expressed as the number of nymphs per five(5) feet of row (5 row feet).

Table 6 shows the number of nymphs detected per 5 row feet fortransgenic cotton event MON 88702 and the negative control DP393,wherein “SEM” is the standard error of the mean and “N” is the number ofplants caged and sampled. The Lygus pressure shown in Table 6 isdetermined relative to the economic threshold for applying insecticideon the cotton field. The economic threshold for applying insecticide oncotton for Lygus control is defined differently depending on the type ofsurvey method used. The conventional economic threshold using the dropcloth method for Lygus lineolaris is three (3) total Lygus (any stage)per 5 row feet. The economic threshold for spraying insecticide oncotton for Lygus hesperus control using the sweep method is fifteen (15)total Lygus (including 4-8 nymphs) per one hundred sweeps. Because ofnatural variation in Lygus pressure in our trials, locations werecategorized as low, medium, or high pressure sites. Low Lygus pressureis defined as being below the economic threshold throughout the season.Medium Lygus pressure is defined as being equal or greater than onetimes the economic threshold, or less than three times the economicthreshold at any time in the season. High Lygus pressure is defined asbeing equal or greater than three times the economic threshold, at anytime in the season.

For transgenic trait efficacy evaluation, total nymphs numbers as adultscaught during sampling cannot be assumed to have developed on thatparticular plot. As can be seen in Table 6, the total number of nymphsdetected in the transgenic event MON 88702 rows was consistently lessthan the total number of nymphs in the untransformed control rows. Thisobservation was consistent regardless of the natural Lygus pressure ineach field throughout the three (3) years. This demonstrates thattransgenic cotton event MON 88702 provides superior resistance to bothLygus lineolaris and Lygus hesperus in cotton field experiments relativeto the untransformed cotton.

TABLE 6 Season long average numbers of Lygus nymphs per 5 row feet forMON 88702 and DP393 in the field. Total Natural Lygus Small Large TotalNymphs Year Location Lygus species Pressure Rating Event N Nymphs NymphsNymphs SEM Year 1 MS Lygus lineolaris High MON 88702 20 6.40 0.40 6.801.44 DP393 40 14.80 1.89 16.69 2.24 AR Lygus lineolaris High MON 8870224 8.10 2.85 10.96 3.20 DP393 48 10.21 2.68 12.89 1.71 LA Lyguslineolaris Low M088702 4 0.23 0.08 0.31 0.11 DP393 8 0.20 0.14 0.33 0.06Year 2 MS Lygus lineolaris High MON 88702 20 5.80 0.38 6.18 1.61 DP39340 10.83 1.36 12.19 1.49 AR Lygus lineolaris Medium MON 88702 24 3.880.54 4.42 0.51 DP393 48 4.46 0.85 5.31 0.46 LA Lygus lineolaris Low MON88702 28 1.64 1.05 2.70 0.63 DP393 56 3.90 1.89 5.79 0.98 AZ Lygushesperus Low MON 88702 36 2.78 1.39 4.17 0.99 DP393 72 2.01 2.92 4.930.67 CA Lygus hesperus Medium MON 88702 24 1.88 5.21 7.08 2.87 DP393 483.33 11.67 15.00 3.80 Year 3 MS Lygus lineolaris High MON 88702 16 6.471.47 7.94 1.51 DP393 16 11.31 6.28 17.59 1.25 AR Lygus lineolaris MediumMON 88702 16 2.22 0.31 2.53 0.43 DP393 16 3.31 0.31 3.63 0.74 LA Lyguslineolaris Medium MON 88702 28 1.54 1.02 2.55 0.48 DP393 28 2.07 1.823.89 0.71 AR Lygus lineolaris Low MON 88702 16 0.33 0.18 0.50 0.18 DP39316 0.70 0.53 1.23 0.17

Example 4 Cotton Event MON 88702 Provides Superior Yield in the FieldUnder Low, Medium, and High Natural Lygus Pressure

This Example demonstrates the ability for transgenic cotton event MON88702 to provide superior yield in fields infested with the Hemipteranpest species Lygus hesperus (Western tarnished plant bug) and Lyguslineolaris (Tarnished plant bug) relative to untransformed cottonplants.

Field trials were conducted in three consecutive years in variouslocations which experienced different levels of natural Lygus pressureas described in Example 3. The two center rows of each eight row plotwere harvested and the yield was measured and expressed in pounds ofseed cotton per acre (lbs/acre).

Table 7 demonstrates the yield of seed cotton per acre for each of thefield locations and is divided based upon the natural Lygus pressureexperienced in each field. The column percent change vs. control showsthe increased percentage of yield on the MON 88702 event relative to theuntransformed DP393 negative control.

TABLE 7 Seed cotton yield for event MON 88702 and untransformed DP393 infields under varying Lygus pressure. Natural Percent Lygus MON changePressure 88702 DP393 vs. Rating Year Location Lygus species (lbs/acre)(lbs/acre) control High 3 MS Lygus lineolaris 2646 1728  53% 2 MS Lyguslineolaris 2101 816 157% 1 MS Lygus lineolaris 2248 1377  63% AR Lyguslineolaris 2206 1303  69% Medium 3 LA Lygus lineolaris 3173 2384  33%,AR Lygus lineolaris 2075 1238  68% 2 AR Lygus lineolaris 2787 1911  46%CA Lygus hesperus 3886 2696  44% Low 3 AR Lygus lineolaris 3109 2872  8%2 LA Lygus lineolaris 2509 2256  11% AZ Lygus hesperus 3490 3128  12% 1LA Lygus lineolaris 2140 2257  −5%

As demonstrated in Table 7, cotton transgenic event MON 88702consistently provided higher yields of cotton under different degrees ofnatural Lygus pressure with the exception of the low pressure year 1 LAtrial. The following year, a yield enhancement of 11% was observed forthis same location under low Lygus pressure. The yield enhancement seenwith event MON 88702 was greater in fields experiencing medium and highLygus pressure. Under medium Lygus pressure, event MON 88702 providedyield increases of thirty-three (33) to sixty-eight (68) percent,depending on the location grown. Under high Lygus pressure, event MON88702 provided yield increases of fifty-three (53) to one hundred seven(157) percent, depending on the location grown.

Example 5 Cotton Event MON 88702 Provides Superior Yield in the FieldUnder Medium and High Natural Lygus Pressure with or without InsecticideApplications

This Example illustrates the superior yield of transgenic cotton eventMON 88702 under medium and high Lygus pressure with or withoutapplication of insecticides when compared to untransformed cotton of thesame variety.

Cotton yield in insecticide spray assays was assessed at six locationsin a single planting season. Eight (8) row plots of cotton were grownusing one hundred twenty (120) seeds treated with Acceleron® (whichcontained fungicides, the insecticide Imidacloprid, and the nematacideThiodicarb) per each thirty (30) foot long row for event MON 88702. Theuntransformed cotton variety DP393 was planted and used as a negativecontrol. Each plot was replicated three (3) times in the field. Three(3) treatment blocks were designed to evaluate the yield of cotton underconditions when the plots were not treated with insecticide to controlLygus or were treated with insecticide to control Lygus. The treatmentblock where no insecticide to control Lygus was applied during theseason is referred to as the “no spray block.” Two treatment blocks inwhich insecticide to control Lygus was applied are referred to as “sprayby entry” and “spray by block.” For spray by entry, the individual entrycontaining MON 88702 or untransformed DP393 is sprayed with insecticidewhen the average of the three (3) reps of that particular entry is atconventional economic threshold for Lygus lineolaris or fifteen (15)Lygus per one hundred (100) sweeps for Lygus hesperus. For spray byblock, the entire block which contains both event MON 88702 anduntransformed DP393 is sprayed with insecticide when the average of thethree (3) reps of the DP393 in that block is at economic threshold forLygus lineolaris or fifteen (15) Lygus per one hundred sweeps for Lygushesperus. Table 8 shows the yield expressed in lbs/acre for the three(3) blocks at each location tested, along with the natural Lyguspressure experienced at each location.

As demonstrated in Table 8, insecticide-untreated andinsecticide-treated event MON 88702 provided greater yield relative tothe corresponding untransformed control DP393. Without insecticidetreatment, the percent increase in yield ranged from 21% to 188%,depending on the location. With spray by entry, the percent increase inyield ranged from 5% to 72%, depending on location. With spray by block,the percent increase in yield ranged from 8% to 47%, depending onlocation. In all locations, whether or not treated with insecticide,transgenic cotton event MON 88702 provided increased yield relative tothe untransformed control.

TABLE 8 Seed cotton yield (lbs/acre) for event MON 88702 anduntransformed DP393 untreated or treated with insecticides. No sprayblock Spray by entry Spray by block Natural Percent MON DP393 PercentMON DP393 Percent Lygus change 88702 Yield change 88702 Yield changePressure MON vs. (# (# vs. (# (# vs. Location Lygus species Rating 88702DP393 DP393 sprays) sprays) DP393 sprays) sprays) DP393 MS Lyguslineolaris High 2993 1558  92% 2848 2435 17% 2986 2341 28% (3) (4) (4)(4) TN Lygus lineolaris High 2049 1447  42% 2758 2636  5% 2844 2622  8%(4) (5) (4) (4) NC Lygus lineolaris High 2693 1047 157% 3888 2259 72%4460 3252 37% (2) (2) (3) (3) AZ Lygus hesperus High 4987 4065  23% 53874530 18% 5017 4566  9% (1) (1) (1) (1) AR Lygus lineolaris Medium 2430843 188% 2465 1955 26% 3330 2258 47% (2) (2) (2) (2) LA Lygus lineolarisMedium 1402 1155  21% 1997 1613 23% 2273 1816 25% (2) (3) (3) (3)

Example 6 Cotton Event MON 88702 Provides Resistance in the Field UnderMedium and High Natural thrips Pressure

This Example illustrates the ability of transgenic cotton event MON88702 to provide excellent resistance to thrips under medium and highnatural thrips pressure when compared to untransformed cotton plants inthe field during a single planting season at multiple field locations.

Eight (8) row plots of cotton were grown using one hundred twenty (120)seeds for event MON 88702 or the untransformed cotton variety DP393 perthirty (30) foot long row (four seeds per row foot) treated withAcceleron® (which contains fungicides, the insecticide Imidacloprid, andthe nematacide Thiodicarb). Each plot was replicated four (4) times inthe field.

Damage to the cotton plants caused by thrips was assessed during the 2-4true leaf stage using a damage rating scale of 0-5. A damage ratingscore of zero (0) equaled no damage and no thrips observed. A damagerating of one (1) corresponded to an indication of thrips being present.A damage rating of two (2) corresponded to minor injury to the terminalbud and leaves. A damage rating of three (3) corresponded to a moderateinjury to the terminal bud and leaves. A damage rating of four (4)corresponded to severe injury to the terminal bud and leaves with somedead plants and aborted terminal buds. A damage rating of five (5)corresponded to plant death, severe stunting, stacked internodes,reduced leaf area and terminal bud abortion of most plants. Because ofnatural variation in thrips pressure in the trials, locations werecategorized as low, medium, or high pressure sites. Low, medium, andhigh natural thrips pressure is defined by the highest damage ratingscore recorded for the untransformed variety at any rating time at thatparticular location. Low thrips pressure corresponds to a damage ratingbelow two (2). Medium thrips pressure corresponds to a damage rating ofequal to or great than two (2) and less than four (4). High thripspressure corresponds to a damage rating of equal to or greater than four(4). Table 9 demonstrates the average thrips damage ratings for threelocations grown in a single planting season.

TABLE 9 Average thrips damage ratings for MON 88702 and the negativecontrol DP393 in 2014. Natural Thrips MON Pressure Location 88702 DP393High MS 1.3 4 Medium TN 0.9 3 Medium VA 0.3 2.7

As demonstrated in Table 9, cotton event MON 88702 provides resistanceagainst thrips (Frankliniella spp). The cotton transgenic event MON88702 consistently had significantly lower damage rating scores relativeto the negative control DP393, regardless of the natural thripspressure.

Example 7 Cotton Event MON 88702 Provides Thrips Resistance in the Fieldwith or without Insecticide Application, and with or without SeedTreatment

This Example illustrates the ability of transgenic cotton event MON88702 to provide superior thrips resistance to multiple thrips species,including Frankliniella fusca (Tobacco thrips), Frankliniella tritici(Flower thrips), Frankliniella occidentalis (Western flower thrips), andSericothrips variabilis (soybean thrips), with or without insecticideapplication and with or without seed treatment when compared to theuntransformed DP393 control in fields during a single growing season.

Four (4) row plots of cotton were grown using one hundred twenty (120)seeds of cotton transgenic event MON 88702 or the untransformed DP393per thirty (30) foot long row (four seeds per row foot). Seeds wereeither treated with Acceleron® (which contains fungicides, theinsecticide Imidacloprid, and the nematacide Thiodicarb), or onlyfungicides. In addition, the plots were either sprayed or not sprayedwith insecticide. The experimental design is shown in Table 10. Forthose plots in which an insecticide was sprayed, a prophylactic foliarapplication of Orthene (acephate) was applied at the one (1) to two (2)true leaf stage. Damage to the cotton plants caused by thrips wasassessed at two different time points (first at the one to two true leafstage and second at the three to four true leaf stage) using a damagerating scale of 0-5 as described previously in Example 6. Two damageratings were performed at each location, with the exception of the TNtrials in which four damage ratings were performed for trial 1 and threedamages ratings were performed for trial 2.

TABLE 10 Experimental design of thrips resistance field trial.Insecticide Event Seed Treatment application DP393 Fungicide OnlyUnsprayed Fungicide Only Sprayed Acceleron ® Unsprayed Acceleron ®Sprayed MON Fungicide Only Unsprayed 88702 Fungicide Only SprayedAcceleron ® Unsprayed Acceleron ® Sprayed

Thrips damage ratings were obtained from multiple sites: VA, MS site 1,MS site 2, GA, MS site 3, LA site 1, LA site 2, SC, and TN. Two trialswere performed in VA, MS site 3, and TN. Tables 11 through 22 show themean thrips damage ratings for cotton transgenic event MON 88702 and theuntransformed control, DP393 for each field site trail, wherein “SEM” isthe standard error of the mean.

TABLE 11 Mean thrips damage ratings from VA, trial 1. Seed InsecticideRating Event Treatment application Date Rating SEM DP393 Fungicide OnlyUnsprayed 26-May 3.50 0.00 Fungicide Only Unsprayed 1-Jun 4.25 0.10Fungicide Only Sprayed 26-May 3.50 0.00 Fungicide Only Sprayed 1-Jun4.00 0.00 Acceleron ® Unsprayed 26-May 2.00 0.00 Acceleron ® Unsprayed1-Jun 3.75 0.00 Acceleron ® Sprayed 26-May 2.06 0.06 Acceleron ® Sprayed1-Jun 2.69 0.06 MON Fungicide Only Unsprayed 26-May 0.50 0.00 88702Fungicide Only Unsprayed 1-Jun 0.19 0.06 Fungicide Only Sprayed 26-May0.31 0.06 Fungicide Only Sprayed 1-Jun 0.25 0.00 Acceleron ® Unsprayed26-May 0.00 0.00 Acceleron ® Unsprayed 1-Jun 0.00 0.00 Acceleron ®Sprayed 26-May 0.00 0.00 Acceleron ® Sprayed 1-Jun 0.00 0.00

TABLE 12 Mean thrips damage ratings from VA, trial 2. Seed InsecticideRating Event Treatment application Date Rating SEM DP393 Fungicide OnlyUnsprayed 1-Jun 4.44 0.06 Fungicide Only Unsprayed 11-Jun 4.19 0.12Fungicide Only Sprayed 1-Jun 4.44 0.06 Fungicide Only Sprayed 11-Jun4.25 0.10 Acceleron ® Unsprayed 1-Jun 3.75 0.00 Acceleron ® Unsprayed11-Jun 4.19 0.06 Acceleron ® Sprayed 1-Jun 3.75 0.14 Acceleron ® Sprayed11-Jun 4.13 0.07 MON Fungicide Only Unsprayed 1-Jun 0.25 0.10 88702Fungicide Only Unsprayed 11-Jun 0.19 0.06 Fungicide Only Sprayed 1-Jun0.31 0.06 Fungicide Only Sprayed 11-Jun 0.13 0.07 Acceleron ® Unsprayed1-Jun 0.06 0.06 Acceleron ® Unsprayed 11-Jun 0.06 0.06 Acceleron ®Sprayed 1-Jun 0.06 0.06 Acceleron ® Sprayed 11-Jun 0.13 0.07

TABLE 13 Mean thrips damage ratings from MS site 1. Seed InsecticideRating Event Treatment application Date Rating SEM DP393 Fungicide OnlyUnsprayed 27-May 3.00 0.20 Fungicide Only Unsprayed 4-Jun 3.75 0.14Fungicide Only Sprayed 27-May 2.88 0.24 Fungicide Only Sprayed 4-Jun3.50 0.00 Acceleron ® Unsprayed 27-May- 2.00 0.41 Acceleron ® Unsprayed4-Jun 2.38 0.24 Acceleron ® Sprayed 27-May 1.75 0.25 Acceleron ® Sprayed4-Jun 2.25 0.14 MON Fungicide Only Unsprayed 27-May 0.88 0.31 88702Fungicide Only Unsprayed 4-Jun 1.13 0.13 Fungicide Only Sprayed 27-May1.25 0.14 Fungicide Only Sprayed 4-Jun 1.38 0.13 Acceleron ® Unsprayed27-May 0.50 0.29 Acceleron ® Unsprayed 4-Jun- 0.75 0.25 Acceleron ®Sprayed 27-May 0.50 0.29 Acceleron ® Sprayed 4-Jun 0.50 0.29

TABLE 14 Mean thrips damage ratings from MS site 2. Seed InsecticideRating Event Treatment application Date Rating SEM DP393 Fungicide OnlyUnsprayed 11-Jun 2.38 0.13 Fungicide Only Unsprayed 17-Jun 3.56 0.06Fungicide Only Sprayed 11-Jun 2.25 0.14 Fungicide Only Sprayed 17-Jun3.38 0.16 Acceleron ® Unsprayed 11-Jun 2.13 0.13 Acceleron ® Unsprayed17-Jun 3.06 0.06 Acceleron ® Sprayed 11-Jun- 2.00 0.00 Acceleron ®Sprayed 17-Jun- 2.25 0.31 MON Fungicide Only Unsprayed 11-Jun 1.50 0.0088702 Fungicide Only Unsprayed 17-Jun 0.06 0.06 Fungicide Only Sprayed11-Jun 1.38 0.13 Fungicide Only Sprayed 17-Jun 0.00 0.00 Acceleron ®Unsprayed 11-Jun 1.25 0.14 Acceleron ® Unsprayed 17-Jun 0.00 0.00Acceleron ® Sprayed 11-Jun 1.13 0.13 Acceleron ® Sprayed 17-Jun 0.000.00

TABLE 15 Mean thrips damage ratings from GA. Seed Insecticide RatingEvent Treatment application Date Rating SEM DP393 Fungicide OnlyUnsprayed 28-May 3.00 0.00 Fungicide Only Unsprayed 4-Jun 3.75 0.25Fungicide Only Sprayed 28-May 3.00 0.00 Fungicide Only Sprayed 4-Jun3.00 0.00 Acceleron ® Unsprayed 28-May 2.25 0.25 Acceleron ® Unsprayed4-Jun 3.00 0.00 Acceleron ® Sprayed 28-May 2.25 0.25 Acceleron ® Sprayed4-Jun- 3.00 0.00 MON Fungicide Only Unsprayed 28-May 1.50 0.29 88702Fungicide Only Unsprayed 4-Jun 2.00 0.00 Fungicide Only Sprayed 28-May1.75 0.25 Fungicide Only Sprayed 4-Jun 1.50 0.29 Acceleron ® Unsprayed28-May 1.50 0.29 Acceleron ® Unsprayed 4-Jun 1.25 0.25 Acceleron ®Sprayed 28-May 1.00 0.00 Acceleron ® Sprayed 4-Jun 1.25 0.25

TABLE 16 Mean thrips damage ratings from MS site 3, trial 1. SeedInsecticide Rating Event Treatment application Date Rating SEM DP393Fungicide Only Unsprayed 28-May 0.75 0.25 Fungicide Only Unsprayed 8-Jun3.00 0.00 Fungicide Only Sprayed 28-May 0.50 0.29 Fungicide Only Sprayed8-Jun 2.25 0.48 Acceleron ® Unsprayed 28-May 0.50 0.29 Acceleron ®Unsprayed 8-Jun 2.25 0.25 Acceleron ® Sprayed 28-May 1.25 0.25Acceleron ® Sprayed 8-Jun 2.25 0.25 MON Fungicide Only Unsprayed 28-May0.50 0.29 88702 Fungicide Only Unsprayed 8-Jun 1.50 0.29 Fungicide OnlySprayed 28-May 0.50 0.29 Fungicide Only Sprayed 8-Jun 1.50 0.29Acceleron ® Unsprayed 28-May 0.50 0.29 Acceleron ® Unsprayed 8-Jun 1.750.25 Acceleron ® Sprayed 28-May 0.50 0.29 Acceleron ® Sprayed 8-Jun 1.500.29

TABLE 17 Mean thrips damage ratings from MS site 3, trial 2. SeedInsecticide Rating Event Treatment application Date Rating SEM DP393Fungicide Only Unsprayed 19-Jun 2.00 0.00 Fungicide Only Unsprayed24-Jun 3.00 0.00 Fungicide Only Sprayed 19-Jun 1.50 0.29 Fungicide OnlySprayed 24-Jun 2.00 0.41 Acceleron ® Unsprayed 19-Jun 1.75 0.25Acceleron ® Unsprayed 24-Jun 2.00 0.00 Acceleron ® Sprayed 19-Jun 1.750.25 Acceleron ® Sprayed 24-Jun 2.00 0.00 MON Fungicide Only Unsprayed19-Jun- 1.25 0.25 88702 Fungicide Only Unsprayed 24-Jun- 1.50 0.29Fungicide Only Sprayed 19-Jun 1.00 0.00 Fungicide Only Sprayed 24-Jun1.50 0.29 Acceleron ® Unsprayed 19-Jun 1.50 0.29 Acceleron ® Unsprayed24-Jun 1.75 0.25 Acceleron ® Sprayed 19-Jun 1.25 0.25 Acceleron ®Sprayed 24-Jun 2.00 0.00

TABLE 18 Mean thrips damage ratings from LA site 1. Seed InsecticideRating Event Treatment application Date Rating SEM DP393 Fungicide OnlyUnsprayed 19-May 3.00 0.00 Fungicide Only Unsprayed 27-May 4.00 0.00Fungicide Only Sprayed 19-May 2.75 0.25 Fungicide Only Sprayed 27-May3.75 0.25 Acceleron ® Unsprayed 19-May 2.00 0.00 Acceleron ® Unsprayed27-May 3.25 0.25 Acceleron ® Sprayed 19-May 2.25 0.25 Acceleron ®Sprayed 27-May 3.00 0.00 MON Fungicide Only Unsprayed 19-May 1.75 0.2588702 Fungicide Only Unsprayed 27-May 2.25 0.25 Fungicide Only Sprayed19-May 1.75 0.25 Fungicide Only Sprayed 27-May 2.25 0.25 Acceleron ®Unsprayed 19-May 1.00 0.00 Acceleron ® Unsprayed 27-May 2.00 0.00Acceleron ® Sprayed 19-May 1.25 0.25 Acceleron ® Sprayed 27-May 2.000.00

TABLE 19 Mean thrips damage ratings from LA (site 2). Insecticide EventSeed Treatment application Rating Date Rating SEM DP393 Fungicide OnlyUnsprayed 19-May 2.00 0.00 Fungicide Only Unsprayed 28-May 3.00 0.00Fungicide Only Sprayed 19-May 1.75 0.25 Fungicide Only Sprayed 28-May2.25 0.25 Acceleron ® Unsprayed 19-May 1.25 0.25 Acceleron ® Unsprayed28-May 2.00 0.00 Acceleron ® Sprayed 19-May 1.67 0.33 Acceleron ®Sprayed 28-May 1.33 0.33 MON Fungicide Only Unsprayed 19-May 1.00 0.0088702 Fungicide Only Unsprayed 28-May 1.25 0.25 Fungicide Only Sprayed19-May 1.50 0.29 Fungicide Only Sprayed 28-May 1.25 0.25 Acceleron ®Unsprayed 19-May 1.00 0.00 Acceleron ® Unsprayed 28-May 1.50 0.29Acceleron ® Sprayed 19-May 1.25 0.25 Acceleron ® Sprayed 28-May 1.250.25

TABLE 20 Mean thrips damage ratings from SC. Insecticide Event SeedTreatment application Rating Date Rating SEM DP393 Fungicide OnlyUnsprayed 21-May 2.25 0.25 Fungicide Only Unsprayed 28-May- 2.00 0.00Fungicide Only Sprayed 21-May 2.00 0.00 Fungicide Only Sprayed 28-May2.00 0.00 Acceleron ® Unsprayed 21-May 2.00 0.00 Acceleron ® Unsprayed28-May 1.75 0.25 Acceleron ® Sprayed 21-May 2.25 0.25 Acceleron ®Sprayed 28-May 2.00 0.00 MON 88702 Fungicide Only Unsprayed 21-May 1.000.00 Fungicide Only Unsprayed 28-May 1.50 0.29 Fungicide Only Sprayed21-May 1.75 0.25 Fungicide Only Sprayed 28-May 1.25 0.25 Acceleron ®Unsprayed 21-May- 1.00 0.00 Acceleron ® Unsprayed 28-May 1.25 0.25Acceleron ® Sprayed 21-May 1.00 0.00 Acceleron ® Sprayed 28-May 1.500.29

TABLE 21 Mean thrips damage ratings from TN, trial 1. Insecticide RatingEvent Seed Treatment application Date Rating SEM DP393 Fungicide OnlyUnsprayed 25-May 3.50 Fungicide Only Unsprayed 28-May 3.60 0.06Fungicide Only Unsprayed 5-Jun 4.03 0.03 Fungicide Only Unsprayed 12-Jun4.03 0.12 Fungicide Only Sprayed 25-May 2.97 0.03 Fungicide Only Sprayed28-May 2.55 0.09 Fungicide Only Sprayed 5-Jun 3.18 0.28 Fungicide OnlySprayed 12-Jun 2.68 0.17 Acceleron ® Unsprayed 25-May 2.05 0.15Acceleron ® Unsprayed 28-May 2.33 0.03 Acceleron ® Unsprayed 5-Jun 2.900.07 Acceleron ® Unsprayed 12-Jun 3.05 0.03 Acceleron ® Sprayed 25-May2.03 0.12 Acceleron ® Sprayed 28-May 1.80 0.04 Acceleron ® Sprayed 5-Jun2.20 0.07 Acceleron ® Sprayed 12-Jun 2.33 0.17 MON 88702 Fungicide OnlyUnsprayed 25-May 1.70 0.10 Fungicide Only Unsprayed 28-May 1.50 0.10Fungicide Only Unsprayed 5-Jun 0.70 0.10 Fungicide Only Unsprayed 12-Jun1.77 0.27 Fungicide Only Sprayed 25-May 1.83 0.13 Fungicide Only Sprayed28-May 1.55 0.10 Fungicide Only Sprayed 5-Jun 0.53 0.08 Fungicide OnlySprayed 12-Jun 0.83 0.09 Acceleron ® Unsprayed 25-May 1.65 0.15Acceleron ® Unsprayed 28-May 1.50 0.10 Acceleron ® Unsprayed 5-Jun- 0.450.15 Acceleron ® Unsprayed 12-Jun 0.75 0.35 Acceleron ® Sprayed 25-May1.68 0.06 Acceleron ® Sprayed 28-May 1.33 0.11 Acceleron ® Sprayed 5-Jun0.33 0.03 Acceleron ® Sprayed 12-Jun 0.40 0.04

TABLE 22 Mean thrips damage ratings from TN, trial 2. Insecticide RatingEvent Seed Treatment application Date Rating SEM DP393 Fungicide OnlyUnsprayed 8-Jun 3.03 0.03 Fungicide Only Unsprayed 11-Jun 3.48 0.23Fungicide Only Unsprayed 15-Jun 3.50 0.04 Fungicide Only Sprayed 8-Jun3.00 0.04 Fungicide Only Sprayed 11-Jun 3.33 0.18 Fungicide Only Sprayed15-Jun 2.85 0.10 Acceleron ® Unsprayed 8-Jun 2.23 0.11 Acceleron ®Unsprayed 11-Jun 2.28 0.11 Acceleron ® Unsprayed 15-Jun 2.40 0.15Acceleron ® Sprayed 8-Jun 2.18 0.08 Acceleron ® Sprayed 11-Jun 2.18 0.13Acceleron ® Sprayed 15-Jun 1.98 0.17 MON 88702 Fungicide Only Unsprayed8-Jun 1.20 0.11 Fungicide Only Unsprayed 11-Jun 0.78 0.09 Fungicide OnlyUnsprayed 15-Jun 0.68 0.09 Fungicide Only Sprayed 8-Jun 1.10 0.13Fungicide Only Sprayed 11-Jun 0.80 0.07 Fungicide Only Sprayed 15-Jun0.50 0.00 Acceleron ® Unsprayed 8-Jun 0.73 0.11 Acceleron ® Unsprayed11-Jun 0.58 0.05 Acceleron ® Unsprayed 15-Jun 0.55 0.06 Acceleron ®Sprayed 8-Jun 0.70 0.04 Acceleron ® Sprayed 11-Jun 0.53 0.05 Acceleron ®Sprayed 15-Jun 0.35 0.03

As demonstrated in Tables 11 through 22, transgenic cotton event MON88702 provides resistance to thrips under field conditions relative tothe same untransformed variety. Transgenic event MON 88702 providedresistance to thrips at each field location when compared to theuntransformed DP393. Resistance to thrips was observed in transgenicevent MON 88702 without insecticidal seed treatment as well as withoutprophylactic foliar insecticide application. The thrips damage ratingsfor unsprayed MON 88702 plants grown from seed only treated withfungicide was lower than that of the untransformed DP393 that was bothgrown from seed treated with insecticide and sprayed with foliarinsecticide. This is particularly salient in the two trials performed inVA (Tables 10 and 11). In many locations such as VA, MS site 2, and GA,the damage ratings for MON 88702 declined from the first data time pointto the next, while the opposite trend was observed for the untransformedDP393, regardless of seed treatment and prophylactic spray. Thripsadults were collected and identified to assess species composition ateach location. The observed species composition is shown in Table 23

TABLE 23 Thrips composition across different locations thrips trials.Frankliniella Frankliniella Frankliniella occidentalis Sericothripsfusca tritici (Western variabilis Date of (Tobacco (Flower Flower(Soybean other Field Location collection Thrips) Thrips) Thrips) Thrips)Thrips MS site 3 28-May 100 0 0 0 0 MS site 3 8-Jun. 100 0 0 0 0 MS site1 28-May 100 0 0 0 0 MS site 1 4-Jun. 78 0 0 22 0 MS site 2 11-Jun. 96 00 4 0 MS site 2 17-Jun. 94 0 0 3 3 LA site 1 27-May 100 0 0 0 0 LA site2 19-May 20 20 60 0 0 LA site 2 28-May 67 0 33 0 0 TN 28-May 91 8 2 0 0TN 5-Jun. 88 5 6 1 0 TN 11-Jun. 88 7 1 5 0 TN 15-Jun. 92 4 0 3 1 GA21-May 64 25 11 0 0 GA 28-May 46 26 26 0 1 GA 4-Jun. 64 21 14 0 1 SC28-May 19 48 22 11 0 SC 3-Jun. 30 35 17 18 0 VA 21-May 48 32 1 18 1 VA1-Jun. 80 13 3 4 1 VA 1-Jun. 83 9 6 1 0 VA 8-Jun. 74 17 9 0 0

Example 8 Cotton Event MON 88702 Provides Resistance to CottonFleahopper (Pseudatomoscelis Seriatus) as Demonstrated in Cage and OpenField Trials in Two Consecutive Years

This example illustrates that transgenic cotton event MON 88702 providesresistance against the Hemipteran insect pest, Pseudatomoscelis seriatus(cotton fleahopper, CFH) when compared to the untransformed negativecontrol, DP393.

Cage trials were performed in the field in TX during the year 1 cottongrowing season and assayed for resistance against cotton fleahopper. Two(2) row plots of cotton were grown using one hundred twenty (120) seedsof cotton transgenic event MON 88702 or the untransformed DP393 negativecontrol per thirty (30) foot long row (four seeds per row foot). Seedswere treated with Acceleron® which contains fungicides, the insecticideImidacloprid, and the nematacide Thiodicarb.

At the second week of squaring, which occurs around thirty five (35) toforty (40) days after planting, ten (10) randomly selected plants (five(5) from each row) were enclosed in a cage (150×228 centimeters) madefrom white solid voile (JoAnne Fabrics, Item Number 8139875). The bottomvoile sleeve was secured to the main plant stem just above the soilsurface and the top sleeve was tied together using a Velcro® tie. Metalpoles were placed at the end of each row and the top of each cage wasattached to a string to keep the cages in an upright position.

Each plant was infested with two pairs of cotton fleahopper male andfemale adults. The adults were released into each individual cage andthen the cage was securely closed ensuring the insects would not escape.The insects were allowed to mate and the plants were kept in the cagefor thirty (30) days. After thirty days, the plants were then cut belowthe cages and moved to a laboratory where the insects were collected foreach plant and counted. Before opening the cage, the plants werevigorously shaken to ensure all of the insects fell off from theirfeeding sites to the base of the cage. Then the cage base was opened.The plant was then thoroughly inspected to recover any remaininginsects. The numbers of insects and their developmental stage wererecorded for each plant. The mean numbers of next generation smallnymphs (prior to 3^(rd) instar), large nymphs (4^(th) and 5^(th)instars) and adults is presented in Table 24, wherein “SEM” is thestandard error of the mean and “N” is the number of plants caged andsampled.

TABLE 24 Average next generation insect counts for cotton event MON88702 and the negative control DP393 infested with CFH in TX, year 1field caging trial. Total Small Large Total Total CFH Event N NymphsNymphs Nymphs Adults CFH SEM MON 88702 8 4.5 2.5 7 3.5 10.5 5.59 DP393 76.43 1.43 7.86 13 20.86 5.23

As can be seen in Table 24, the total number of cotton fleahopperinsects recovered from the cages for cotton event MON 88702 wasapproximately half the number found in the untransformed DP393 control.Of particular importance is the number of next generation adultsrecorded. The untransformed DP393 had approximately 4-fold more adultscompared to event MON 88702, suggesting that over the course ofdevelopment, fewer nymphs reach adulthood due to the expressed insecttoxin, TIC834_16 in MON 88702. Cotton fleahoppers overwinter in the eggstage in various weed hosts including croton, evening primrose,silverleaf nightshade and lanceleaf sage. Eggs hatch in the spring withCotton fleahoppers infesting available weed hosts. Once cotton begins tosquare, adult Cotton fleahoppers can and often do move into thesefields. It usually takes a generation in the earlier cotton fields toproduce economically damaging numbers. Reducing the number of adultCotton fleahoppers in this earlier generation can potentially reduce theyield and economic impact caused by the next generation of Cottonfleahopper.

In year 2, the cotton fleahopper trials were conducted in a greenhouse.Twelve (12) plants of transgenic cotton event MON 88702 and theuntransformed DP393 were grown in the greenhouse and were caged in asimilar manner as described above. Infestation of cotton fleahopper andcounts of small nymphs, large nymphs, and adults were also performed aspreviously described except that three pairs of adults were released ineach cage. Table 25 shows the mean numbers of next generation smallnymphs, large nymphs, and adults found for MON 88702 and DP393, wherein“SEM” is the standard error of the mean and “N” is the number of plantscaged and sampled.

TABLE 25 Average next generation insect counts for cotton event MON88702 and the negative control DP393 infested with CFH in TX, year 2greenhouse caging trial. Total Small Large Total CFH Event N NymphsNymphs Adults CFH SEM MON 88702 12 17.33 7.50 14.67 39.50 9.33 DP393 1018.10 6.60 42.10 66.80 7.96

As demonstrated in Table 25, the total number of cotton fleahopperspresent on MON 88702 was less than that of the negative control, DP393.Consistent with the previous year's field cage trials, the number ofadult cotton fleahopper associated with the negative control was muchhigher (approximately 3-fold) than that of MON 88702.

In the year 2 growing season, field trials were also conducted againstcotton fleahopper in AZ and TX. Eight (8) row plots of cotton were grownusing one hundred twenty (120) seeds of cotton transgenic event MON88702 or the untransformed DP393 per thirty (30) foot long row (fourseeds per row foot). Seeds were treated with Acceleron® (which containsfungicides, the insecticide Imidacloprid, and the nematacideThiodicarb). There were four (4) reps of each entry at both locations.

Cotton fleahoppers were collected using the sweep net at the AZ locationand the beat bucket at the TX location. The sweep net method essentiallyconsists of sweeping through the top of the canopy for a specifiednumber of sweeps and then counting the number of insects afterseparating for stages. A total of twenty (20) sweeps were conducted foreach plot. The sweeps were conducted in two (2) runs of ten (10) sweepseach. The first run was performed between rows two and three. The secondrun was performed between rows six and seven. Insects trapped in thenets were put in pre-labeled bags and brought back to the lab forcounting. The mean number of cotton fleahopper nymphs for MON 88702 andthe untransformed control DP393 was determined for each site. In thebeat bucket method, plants were shaken into a 5-gallon bucket ensuringall nymphs come off of the plant and all bucket contents were put inprelabeled zip lock bags. Three (3) runs of ten (10) plants samplingeach with a total of thirty (30) plants sampled per plot per time.

In addition to cotton fleahopper counts, each plot was assessed foryield. Bolls were harvested from rows four (4) and five (5) and theaverage seed cotton yield was determined and expressed as seed cottonyield/acre. Table 26 shows the mean number of cotton fleahopper nymphsand the mean yield for MON 88702 and the untransformed control DP393 foreach site wherein “SEM” is the standard error of the mean and “N” is thenumber of plants caged and sampled.

TABLE 26 Mean number of CFH nymphs and mean seed cotton yield in year 2open field trials. CFH Nymphs Yield/acre Location Event N Mean SEM NMean SEM Maricopa, AZ MON 88702 28 8.29 1.73 4 5044.88 90.97 DP393 289.29 1.68 4 4525.13 115.97 Corpus Christi, TX MON 88702 32 3.47 1.07 42727.75 35.69 DP393 32 7.59 0.69 4 2621.25 163.47

Table 26 shows that the mean number of cotton fleahopper nymphs waslower for MON 88702 than in untransformed DP393. In addition, the meancotton yield was higher in MON 88702 than in the untransformed DP393.

As demonstrated in Tables 24-26, cotton event MON 88702 providesresistance to cotton fleahopper in both cage trials and in the field andprovides an increase in cotton yield.

Example 9 Cotton Event MON 88702 Provides Resistance to Verde Plant Bug(Creontiades Signatus) as Demonstrated in Cage Trials

This Example illustrates that transgenic cotton event MON 88702 providesresistance against the hemipteran insect pest Creontiades signatusDistant (Verde plant bug) in cage trials performed in TX when comparedto the untransformed negative control DP393.

Verde plant bug nymphs were collected from plants in the field andbrought back to the laboratory for rearing. Once the adults reachedreproductive maturity (approximately ten (10) days after emergence),they were used to infest cotton event MON 88702 or the untransformedcontrol DP393 in caged cotton plants.

Eight (8) row plots of cotton were grown using one hundred twenty (120)seeds of cotton transgenic event MON 88702 or the untransformed DP393per thirty (30) foot long row (four (4) seeds per row foot). Seeds weretreated with Acceleron® (which contains fungicides, the insecticideImidacloprid, and the nematacide Thiodicarb). Four (4) replicate plotswere grown per event. Twelve (12) plants for each event were randomlyselected for caging. Any insects on the selected plants were removedprior to caging. Plants were caged at least a week before peak bloomingas previously described in Example 2. Each plant was infested with twopairs of male and female Verde plant bug. The insects were allowed tooviposit and develop on each plant for four (4) weeks after infestation.Eight (8) to ten (10) bolls were harvested from each plant and examinedfor damage to the locules, as well as interior and exterior puncturesmade by the Verde plant bugs.

Table 27 shows the mean damaged locules from the harvested bolls,wherein “SEM” is the standard error of the mean and “N” is the number ofplants caged and sampled. As can be seen in Table 27, there were lessdamaged locules in MON 88702 event plants than in the untransformedcontrol DP393.

TABLE 27 Mean damaged locules per boll per plant from plants infestedwith Verde plant bug. Mean Damaged Event N Locules SEM MON 88702 11 0.420.09 DP393 11 0.89 0.16

Table 28 shows the mean exterior and interior punctures wherein “SEM” isthe standard error of the mean and “N” is the number of plants caged andsampled. As can be seen in Table 28, the mean number of exterior andinterior punctures in the bolls was lower for event MON 88702 than inthe untransformed control DP393.

TABLE 28 Mean exterior and interior puncture per boll from plantsinfested with Verde plant bug. Exterior Interior Event N Punctures SEMPunctures SEM MON 88702 11 21.53 1.74 2.47 0.38 DP393 11 29.22 3.46 6.551.53

As demonstrated in Tables 27 and 28, cotton event MON 88702 demonstratesresistance against Verde plant bug as evidenced in the lower amount oflocule damage as well as fewer exterior and interior punctures whencompared to the untransformed negative control DP393.

Example 10 Cotton Event MON 88702 Provides Consistent Yield, SimilarAgronomics, and Similar Fiber Quality in the Field when Compared toUntransformed DP393

This Example demonstrates that transgenic cotton event MON 88702provides consistent yields in the field.

Cotton event MON 88702 was compared to the untransformed DP393 in thefield over two years of assessment in two consecutive years to assessany difference in yield under conditions in which insect infestation wascontrolled using insecticidal sprays against both non-Lygus and Lyguspests. Two (2) row plots of cotton were grown using one hundred sixty(160) seeds of cotton transgenic event MON 88702 or the untransformedDP393 negative control per forty (40) foot long row (four (4) seeds perrow foot). Seeds were treated with Acceleron® which contains fungicides,the insecticide Imidacloprid, and the nematacide Thiodicarb. Each plotwas replicated four (4) times. Insecticidal sprays were applied asneeded to control both Lygus and non-Lygus insect pests to minimize anypotential damage to the plots.

At the end of the season, the growth was terminated by usingcommercially available defoliants and boll openers. Defoliants used inthis manner are often referred to as “harvest aids.” Removing the leavesprior to harvest provides several advantages. For example, removing theleaves before harvest increases the air movement through the crop canopywhich facilitates quicker drying and prevents boll rot. This processalso allows the picker to begin earlier in the day and provides for afaster and more efficient picker operation. By reducing moisture moreeffectively, the storage of the bolls in modules is greatly improved.Removing the leaves also eliminates a main source of stain and trashwhich provides a better lint grade. Boll openers facilitate the openingof mature bolls which permits harvesting operations to start severaldays earlier, increasing the percentage of the crop harvested during thefirst picking, and makes picking an once-over operation in many fields.Defoliants and boll openers were applied when sixty (60) percent of thebolls were open in the field.

To assess yield, both rows of each two (2) row plot were harvested andthe yield recorded as “seed cotton in pounds per acre”. The yieldresults are presented in Table 29.

TABLE 29 Yield of seed cotton in pounds per acre from year 1 and year 2field harvests. Yield Yield Event year 1 year 2 MON 88702 3600.1 3855.4DP393 3585.1 3854.0

As demonstrated in Table 29 above, both MON 88702 and untransformedDP393 provided similar yields of cotton in the field. This demonstratesthat cotton event MON 88702 in expressing the insect toxin TIC834_16does not experience a yield drag due to expression of the transgene. Infact, as shown in Examples 4 and 5, cotton event MON 88702 provides asignificant yield advantage with or without insecticide applicationunder conditions of Lygus pressure.

The plants were also assessed for any phenotypic differences in bothyear 1 and year 2. Examples of phenotypic differences include earlyvigor score (EVS), number of nodes, plant height, and maturity (numberof weeks). Early vigor score is determined at approximately ten (10) tofourteen (14) days after the plants have emerged. The score is a visualrating used to determine if the plot has full yield potential based uponthe emergence of the plants in the plot. The plots are rated using ascale of one to five. A rating of four or five indicates low vigor,while a rating of one corresponds to high vigor. Table 30 shows thephenotypic values between MON 88702 and the negative control DP393 forearly vigor, node number, plant height and plant maturity. Each of thevalues are expressed as an average. Comparisons were performed for eachgrowing season. Those values indicated by “*” represent a significantdifference (p=0.05) compared to the control within the growing season.

TABLE 30 Phenotypic characteristics of MON 88702 and untransformed DP393grown in the field in year 1 and year 2. Node Plant Year Event EVS #Height Maturity 1 MON 88702 2.6 15.8 32.5* 13.7 DP393 2.7 15.4 34.3 14.52 MON 88702 1.9 14.8 32.2 13.6 DP393 2.0 15.1 32.5 13.9

As can be seen in Table 30, cotton transgenic event MON 88702demonstrated similar phenotypic characteristics as the untransformedDP393. A small difference was observed with respect to plant height in2013, however the following year both MON 88702 and DP393 were aroundthe same average height. With respect to early vigor stand, number ofnodes and maturity, both MON 88702 and DP393 were not significantlydifferent across growing seasons.

Twenty five (25) bolls per plot were collected prior to harvest forassessment of fiber quality and other characteristics. Bolls werecollected from a representative spot in the planting row. All bolls wereharvested from the selected plants to prevent the introduction of anybias in the collection due to size or other characteristics. Fiberquality characteristics such as fiber strength, fiber length, lengthuniformity and micronaire were determined from the harvested bolls.Fiber length was measured in inches.

Fiber strength measurement is made by clamping and breaking a bundle offibers with a ⅛-inch spacing between the clamp jaws. Results arereported in terms of grams per tex to the nearest tenth. A tex unit isequal to the weight in grams of one thousand meters of fiber. Therefore,the strength reported is the force in grams required to break a bundleof fibers one tex unit in size. Table 31 shows a general description andcorresponding strength measurements in grams per tex.

TABLE 31 Fiber strength description and corresponding strength measure.Strength Description (grams per tex) Weak ≤23.0 Intermediate 24.0-25.0Average 26.0-28.0 Strong 29.0-30.0 Very Strong ≥31.0

Length uniformity is a three-digit number that is a measure of thedegree of uniformity of the fibers in a sample to the nearest tenth.Table 32 shows the length uniformity description and correspondinglength uniformity score.

TABLE 32 Length uniformity description and score. Length DescriptionUniformity Very Low ≤76.5 Low 76.5-79.4 Average 79.5-82.4 High 82.5-85.4Very High ≥85.4

Micronaire is a measure of fiber fineness and maturity. An airflowinstrument is used to measure the air permeability of a constant mass ofcotton fibers compressed to a fixed volume. The volume of airflowthrough a specimen of cotton fibers is expressed as a micronaire.Cottons with micronaire measurements between 3.7 and 4.2 are consideredin the premium range of micronaire. Cottons within the micronaire rangesof 3.5-3.6 or 4.3-4.9 are considered base quality, while cottons above4.9 or below 3.5 are in the discount ranges. Micronaire measurements canbe influenced during the growing period by environmental conditions suchas moisture, temperature, sunlight, plant nutrients, and extremes inplant or boll population. Favorable growing conditions result in fullymature fibers with premium range micronaire readings. Unfavorableconditions, such as lack of moisture, early freeze, or any otherconditions that interrupt plant processes, will result in immaturefibers and low micronaire measurements. High micronaire cotton is causedby such things as abnormally warm temperatures during boll maturation,or poor boll set leading to excessive availability of carbohydrates andover-maturing of fibers. Fiber fineness affects processing performanceand the quality of the end-product in several ways. In the opening,cleaning, and carding processes, low micronaire or fine-fiber cottonsrequire slower processing speeds to prevent damage to the fibers. Yarnsmade from finer fiber result in more fibers per cross section, which inturn produces stronger yarns. High micronaire or coarse fibers are notsuitable for fine yarns since the result would be fewer fibers per crosssection, which would reduce the yarn strength. Micronaire and maturityare highly correlated within a cotton variety. Dye absorbency andretention varies with the maturity of the fibers. Low maturity fibershave poor dye absorbency and retention while higher micronaire fibershave good absorbency and retention.

Table 33 shows the fiber quality measures determined for both MON 88702and the untransformed negative control DP393 for two consecutive growingseasons, year 1 and year 2. Those values indicated by “*” represent asignificant difference (p=0.05) compared to the control within thegrowing season.

TABLE 33 Fiber quality of MON 88702 and DP393. Year Event LengthStrength Uniformity Micronaire 1 MON 88702 1.19 32.9 85.6 4.5 DP393 1.1933.6 85.5 4.8 2 MON 88702 1.15 32.1* 84.6 4.83 DP393 1.13 34.7 84.4 5.04

As can be seen in Table 33, both event MON 88702 and the untransformedDP393 produced fiber of very similar qualities in both the 2013 and 2014growing season. Event MON 88702 produced fiber with a similar length asthe negative control. Both MON 88702 and DP393 produce very strong fiberwith high to very high uniformity. The micronaire measures were notsignificantly different between MON 88702 and DP393. These resultdemonstrate that fiber quality is not adversely affected by theexpression of the transgene cassette in MON 88702.

Example 11 Cotton Event MON 88702 Event Specific Endpoint TAQMAN® andZygosity Assays

The following Example describes methods useful in identifying thepresence of event MON 88702 in a cotton sample. A pair of PCR primersand a probe were designed for the purpose of identifying the uniquejunction formed between the cotton genomic DNA and the inserted DNA ofevent MON 88702 in an event specific endpoint TAQMAN® PCR. Examples ofconditions utilized for identifying the presence of MON 88702 in acotton sample in an event specific endpoint TAQMAN® PCR are described inTable 34 and Table 35.

The sequence of the oligonucleotide forward primer SQ21940 (SEQ IDNO:11) is identical to the nucleotide sequence corresponding topositions 4720 to 4744 of SEQ ID NO:10. The sequence of theoligonucleotide reverse primer SQ-50210 (SEQ ID NO:12) is identical tothe reverse compliment of the nucleotide sequence corresponding topositions 4803 to 4826 of SEQ ID NO:10. The sequence of theoligonucleotide probe PB10344 (SEQ ID NO:13) is identical to thenucleotide sequence corresponding to positions 4745 to 4766 of SEQ IDNO: 10. The primers SQ21940 (SEQ ID NO:11) and SQ-50210 (SEQ ID NO:12)with probe PB10344 (SEQ ID NO:13), which may be fluorescently labeled(e.g., a 6FAM™ fluorescent label), can be used in an endpoint TAQMAN®PCR assay to identify the presence of DNA derived from event MON 88702in a sample.

In addition to SQ21940 (SEQ ID NO:11), SQ-50210 (SEQ ID NO:12) andPB10344 (SEQ ID NO:13), it should be apparent to persons skilled in theart that other primers and/or probes can be designed to either amplifyand/or hybridize to sequences within SEQ ID NO:10 which are unique to,and useful for, detecting the presence of DNA derived from event MON88702 in a sample.

Following standard molecular biology laboratory practices, PCR assaysfor event identification were developed for detection of event MON 88702in a sample. Parameters of either a standard PCR assay or a TAQMAN® PCRassay were optimized with each set of primer pairs and probes (e.g.,probes labeled with a fluorescent tag such as 6FAM™) used to detect thepresence of DNA derived from event MON 88702 in a sample. A control forthe PCR reaction includes internal control primers and an internalcontrol probe (e.g., VIC®-labeled) specific to a Gossypium hirsutumfiber-specific acyl carrier protein (ACP1) gene (GenBank AccessionU48777), and are primers SQ22496 (SEQ ID NO:14), SQ22497 (SEQ ID NO:15),and VIC® labeled probe PB13032 (SEQ ID NO:17).

Generally, the parameters which were optimized for detection of eventMON 88702 in a sample included primer and probe concentration, amount oftemplated DNA, and PCR amplification cycling parameters. The controlsfor this analysis include a positive control from cotton containingevent MON 88702 DNA, a negative control from non-transgenic cotton, anda negative control that contains no template DNA.

TABLE 34 Cotton event MON 88702 event specific endpoint TAQMAN ® PCRreaction components. Stock Final Concentration Volume Concentration StepReagent (μM) (μl) (μM) Comments Reaction volume 5 1 18 megohm water 0.28Adjust for final volume 2 2X Master Mix 2.5 1X final concentration 3Event Specific 100 0.05 1 Primer SQ-50210 4 Event Specific 100 0.05 1Primer SQ21940 5 Event Specific 100 0.01 0.2 Probe is light sensitive6FAM ™ probe PB10344 6 Internal Control 100 0.05 1 Primer SQ22497 7Internal Control 100 0.05 1 Primer SQ22496 8 Internal Control 100 0.010.2 Probe is light sensitive VIC ® probe PB13032 9 Extracted DNA 2(template): Leaf Samples to be analyzed Negative control (non-transgenicDNA) Negative water control (No template control) Positive Qualitativecontrol(s) MON 88702 DNA

TABLE 35 Endpoint TAQMAN ® thermocycler conditions Step Cycle No. No.Settings 1  1 95° C. 20 seconds 2 10 95° C. 3 seconds 2 10 64° C.-1°C./Cycle 20 seconds 3 30 95° C. 3 seconds 3 30 54° C. 20 seconds 4  110° C. 20 Forever

A zygosity assay is useful for determining if a plant comprising anevent is homozygous for the event DNA (i.e., comprising the exogenousDNA in the same location on each chromosome of a chromosomal pair),heterozygous for the event DNA (i.e., comprising the exogenous DNA ononly one chromosome of a chromosomal pair), or wild-type (i.e., null forthe event DNA). An endpoint TAQMAN® thermal amplification method wasused to develop a zygosity assay for event MON 88702. Examples ofconditions that may be used in an event specific zygosity TAQMAN® PCRare provide in Tables 36 and 37. For this assay, three primers and twoprobes were mixed together with the sample. The DNA primers used in thezygosity assay were primers SQ50844 (SEQ ID NO:17), SQ50843 (SEQ IDNO:19), and SQ50842 (SEQ ID NO:20). The probes used in the zygosityassay were 6FAM™-labeled probe PB50279 (SEQ ID NO:18) and VIC®-labeledprobe PB50278 (SEQ ID NO:21). Primers SQ50844 (SEQ ID NO:17) and SQ50843(SEQ ID NO:19) and the probe PB50279 (SEQ ID NO:18) (6FAM™-labeled) arediagnostic for event MON 88702 DNA. Primers SQ50842 (SEQ ID NO:20) andSQ50843 (SEQ ID NO:19) and the VIC®-labeled probe PB50278 (SEQ ID NO:21)are diagnostic when there is no copy of MON 88702; i.e., the wild typeallele.

When the three primers and two probes are mixed together in a PCRreaction with DNA extracted from a plant heterozygous for event MON88702, there is a fluorescent signal from both the 6FAM™-labeled probePB50279 (SEQ ID NO:18) and the VIC®-labeled probe PB50278 (SEQ ID NO:21)which is indicative of and diagnostic for a plant heterozygous for eventMON 88702. When the three primers and the two probes are mixed togetherin a PCR reaction with DNA extracted from a plant which is null forevent MON 88702 (i.e., the wild-type), there is a fluorescent signalfrom only the VIC®-labeled probe PB50278 (SEQ ID NO:21) which isindicative of and diagnostic for a plant null for event MON 88702. Thetemplate DNA samples and controls for this analysis were a positivecontrol from cotton containing event MON 88702 DNA (from both a knownhomozygous and a known heterozygous sample), a negative control fromnon-transgenic cotton and a negative control that contains no templateDNA.

TABLE 36 Cotton event MON 88702 zygosity TAQMAN ® PCR Stock FinalConcen- Concen- tration Volume tration Step Reagent (μM) (μl) (μM)Comments Reaction Volume 5 1 18 megohm water 0.33 adjust for finalvolume 2 2X Master Mix 2.5 1X final concentration of buffer 3 SQ50842100 0.05 1 4 SQ50843 100 0.05 1 5 PB50278 100 0.01 0.2 6 SQ50844 1000.05 1 7 PB50279 100 0.01 0.2 8 Extracted DNA 2 (template): Leaf Samplesto be analyzed Negative control (non-transgenic DNA) Negative watercontrol (No template control) Positive Qualitative control(s) MON 88702DNA

TABLE 37 Zygosity TAQMAN ® thermocycler conditions Step Cycle No. No.Settings 1  1 95° C. 20 seconds 2 40 95° C. 3 seconds 2 40 60° C. 20seconds 4  1 10° C. 20 Forever

Example 12 Identification of Cotton Event MON 88702 in any MON 88702Breeding Event

The following Example describes how one may identify the MON 88702 eventwithin progeny of any breeding activity using cotton event MON 88702.

DNA primer pairs are used to produce an amplicon diagnostic for cottonevent MON 88702. An amplicon diagnostic for MON 88702 comprises at leastone junction sequence, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6 ([1], [2], [3], [4], [5], and [6],respectively in FIG. 1a ). SEQ ID NO:1 is a twenty (20) nucleotidesequence representing the 5′ junction regions of cotton genomic DNA andthe integrated transgenic expression cassette. SEQ ID NO:1 is positionedin SEQ ID NO:10 at nucleotide position 1642-1661. SEQ ID NO:2 is atwenty (20) nucleotide sequence representing the 3′ junction regions ofcotton genomic DNA and the integrated transgenic expression cassette.SEQ ID NO:2 is positioned in SEQ ID NO:10 at nucleotide position4785-4804. SEQ ID NO:3 is a sixty (60) nucleotide sequence representingthe 5′ junction regions of cotton genomic DNA and the integratedtransgenic expression cassette. SEQ ID NO:3 is positioned in SEQ IDNO:10 at nucleotide position 1622-1681. SEQ ID NO:4 is a sixty (60)nucleotide sequence representing the 3′ junction regions of cottongenomic DNA and the integrated transgenic expression cassette. SEQ IDNO:4 is positioned in SEQ ID NO:10 at nucleotide position 4765-4824. SEQID NO:5 is a one hundred nucleotide sequence representing the 5′junction regions of cotton genomic DNA and the integrated transgenicexpression cassette. SEQ ID NO:5 is positioned in SEQ ID NO:10 atnucleotide position 1602-1701. SEQ ID NO:6 is a one hundred nucleotidesequence representing the 3′ junction regions of cotton genomic DNA andthe integrated transgenic expression cassette. SEQ ID NO:6 is positionedin SEQ ID NO:10 at nucleotide position 1602-1701.

Primer pairs that will produce an amplicon diagnostic for event MON88702 include primer pairs based upon the flanking sequences and theinserted TIC834_16 expression cassette. To acquire a diagnostic ampliconin which at least eleven nucleotides of SEQ ID NO:1 is found, one woulddesign a forward primer based upon SEQ ID NO:7 from bases 1 through 1651and a reverse primer based upon SEQ ID NO:9. To acquire a diagnosticamplicon in which at least eleven nucleotides of SEQ ID NO:2 is found,one would design a forward primer based upon SEQ ID NO:9 and a reverseprimer based upon SEQ ID NO:8 from bases 1 through 2010. To acquire adiagnostic amplicon in which at least thirty one nucleotides of SEQ IDNO:3 is found, one would design a forward primer based upon SEQ ID NO:7from bases 1 through 1651 and a reverse primer based upon SEQ ID NO:9.To acquire a diagnostic amplicon in which at least thirty onenucleotides of SEQ ID NO:4 is found, one would design a forward primerbased upon SEQ ID NO:9 and a reverse primer based upon SEQ ID NO:8 frombases 1 through 2010. To acquire a diagnostic amplicon in which at leastfifty one nucleotides of SEQ ID NO:5 is found, one would design aforward primer based upon SEQ ID NO:7 from bases 1 through 1651 and areverse primer based upon SEQ ID NO:9. To acquire a diagnostic ampliconin which at least fifty one nucleotides of SEQ ID NO:6 is found, onewould design a forward primer based upon SEQ ID NO:9 and a reverseprimer based upon SEQ ID NO:8 from bases 1 through 2010.

For practical purposes, one should design primers which produceamplicons of a limited size range, preferably between 200 to 1000 bases.Smaller sized amplicons in general are more reliably produced in PCRreactions, allow for shorter cycle times, and can be easily separatedand visualized on agarose gels or adapted for use in endpointTAQMAN®-like assays. In addition, amplicons produced using said primerpairs can be cloned into vectors, propagated, isolated and sequenced, orcan be sequenced directly with methods well established in the art. Anyprimer pair derived from the combination of SEQ ID NO:7 and SEQ ID NO:9,or the combination of SEQ ID NO:8 and SEQ ID NO:9 that are useful in aDNA amplification method to produce an amplicon diagnostic for MON 88702or progeny thereof is an aspect of the present invention. Any singleisolated DNA polynucleotide primer molecule comprising at least elevencontiguous nucleotides of SEQ ID NO:7, or its complement that is usefulin a DNA amplification method to produce an amplicon diagnostic for MON88702 or progeny thereof is an aspect of the present invention. Anysingle isolated DNA polynucleotide primer molecule comprising at leasteleven contiguous nucleotides of SEQ ID NO:8, or its complement that isuseful in a DNA amplification method to produce an amplicon diagnosticfor MON 88702 or progeny thereof is an aspect of the present invention.Any single isolated DNA polynucleotide primer molecule comprising atleast eleven contiguous nucleotides of SEQ ID NO:9, or its complementthat is useful in a DNA amplification method to produce an amplicondiagnostic for MON 88702 or progeny thereof is an aspect of the presentinvention.

An example of the amplification conditions for this analysis isillustrated in Tables 34 and 35 of the previous Example. However, anymodification of these methods or the use of DNA primers homologous orcomplementary to SEQ ID NO:6 or SEQ ID NO:7, or DNA sequences of thegenetic elements contained in the transgene insert (SEQ ID NO:9) of MON88702 that produce an amplicon diagnostic for MON 88702, is within theart. A diagnostic amplicon comprises a DNA molecule homologous orcomplementary to at least one transgene/genomic junction DNA (SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ IDNO:6), or a substantial portion thereof.

An analysis for event MON 88702 plant tissue sample should include apositive tissue control from event MON 88702, a negative control from acotton plant that is not event MON 88702 (for example, but not limitedto DP393) and a negative control that contains no cotton genomic DNA. Aprimer pair will amplify an endogenous cotton DNA molecule and willserve as an internal control for the DNA amplification conditions.Additional primer sequences can be selected from SEQ ID NO: 7, SEQ IDNO: 8, or SEQ ID NO: 9 by those skilled in the art of DNA amplificationmethods, and conditions selected for the production of an amplicon bythe methods shown in Table 34 and Table 35 may differ, but result in anamplicon diagnostic for event MON 88702 DNA. The use of these DNA primersequences with modifications to the methods of Table 34 and Table 35 arewithin the scope of the invention. The amplicon produced by at least oneDNA primer sequence derived from SEQ ID NO:7, SEQ ID NO:8, or SEQ IDNO:9 that is diagnostic for MON 88702 is an aspect of the invention.

DNA detection kits that contain at least one DNA primer of sufficientlength of contiguous nucleotides derived from SEQ ID NO:7, SEQ ID NO:8,or SEQ ID NO:9, that when used in a DNA amplification method, produces adiagnostic amplicon for MON 88702 or its progeny is an aspect of theinvention. A cotton plant or seed, wherein its genome will produce anamplicon diagnostic for MON 88702 when tested in a DNA amplificationmethod is an aspect of the invention. The assay for the MON 88702amplicon can be performed by using an Applied Biosystems GeneAmp PCRSystem 9700, Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700,Eppendorf Mastercycler Gradient thermocycler or any other amplificationsystem that can be used to produce an amplicon diagnostic of MON 87702as shown in Table 35.

Example 13 Crossing of Plants Containing Event MON 87702

To produce cotton plants or plant parts thereof which comprise enhancedagronomic, insecticidal, or herbicidal properties, cotton plantscontaining MON 87702 are crossed with cotton plants containingpotentially any other cotton event or combination thereof and phenotypesare evaluated to determine the resulting properties of the progenyplants. Properties conferred to progeny plants resulting from such plantbreeding can extend beyond the Hemipteran and Thysanopteran resistanceof MON 87702, including, but not limited to above-ground pest control,herbicide tolerance, nematicidal properties, drought resistance, virusresistance, anti-fungal control, bacteria resistance, male sterility,cold tolerance, salt tolerance, and increased yield. Examples oftransgenic events with improved agronomic traits are well known in theart. The following is a non-limiting list of possible transgenic cottonlines which can be used in breeding with MON 87702 to confer enhancedproperties in cotton plants, plant parts, seed or commodity product:19-51A (DD-04951A-7), BXN, MON 1445 (MON-01445-2), MON 88701(MON-88701-3), MON 88913 (MON-88913-8), GHB614 (BCS-GH002-5),DAS-81910-7 (DAS-81910-7), GHB119 (BCS-GH005-8), LLCotton25(ACS-GH0013), EE-GH1, EE-GH3, pDAB4468.18.07.1, pDAB4468.19.10.3,281-24-236 (DAS-24236-5), 3006-210-23 (DAS-21023-5), COT102(SYN-IR102-7), COT67B (SYN-IR67B-1), Event-1, MON 531 (MON-00531-6),MON15985 (MON-15985-7), EE-GHS, EE-GH6, COT202, COT203, A26-5, 31807,31808, T303-3 (BCS-GH003-6), and T304-40 (BCS-GH004-7).

All publications and published patent documents cited in thisspecification, and which are material to the invention, are incorporatedherein by reference to the same extent as if each individual publicationor patent application was specifically and individually indicated to beincorporated by reference.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

1. A recombinant DNA molecule comprising a nucleotide sequence having atleast 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, and complete complementthereof.
 2. The recombinant DNA molecule of claim 1, wherein therecombinant DNA molecule is from cotton event MON 88702, arepresentative sample of seed comprising said event having beendeposited under ATCC Accession No. PTA-122520. 3.-7. (canceled)
 8. Acotton plant, cotton plant part, or cotton cell thereof comprising therecombinant DNA molecule of claim
 1. 9. A cotton plant, cotton plantpart, or cotton cell thereof of claim 8, wherein said cotton plant, orcotton plant part, or cotton cell is insecticidal when provided in thediet of a Hemipteran or Thysanopteran insect pest.
 10. The cotton plant,cotton plant part, or cotton cell thereof of claim 9, wherein theHemipteran insect pest is selected from the group consisting of Lygushesperus, Lygus lineolaris, Creontiades signatus, and Pseudatomoscelisseriatus.
 11. The cotton plant, cotton plant part, or cotton cellthereof of claim 9, wherein the Thysanopteran insect pest is selectedfrom the group consisting of Frankliniella spp and Sericothripsvariabilis.
 12. The cotton plant, cotton plant part, or cotton cellthereof of claim 8, wherein the cotton plant is further defined as aprogeny of any generation of a cotton plant comprising the cotton eventMON
 88702. 13.-16. (canceled)
 17. A cotton seed comprising a detectableamount of the nucleotide sequence of claim
 1. 18. A nonliving cottonplant material comprising a detectable amount of the recombinant DNAmolecule of claim
 1. 19. A microorganism comprising a detectable amountof the recombinant DNA molecule of claim
 1. 20. The microorganism ofclaim 19, wherein the microorganism is a plant cell.
 21. A cottoncommodity product comprising the recombinant DNA molecule of claim 1.22. The cotton commodity product of claim 21, further defined as acommodity product selected from the group consisting of whole or processcotton seeds, cotton fiber, cotton oil and derivatives of cotton oil,cotton protein, cotton meal, animal feed comprising cotton, papercomprising cotton, cotton biomass, candle wicks, cotton string, cottonrope, cotton balls, cotton batting, cotton fuel products, and cottoncellulose products. 23.-27. (canceled)
 28. The recombinant DNA moleculeof claim 1, wherein the recombinant DNA molecule comprises a nucleotidesequence having at least 99% sequence identity to a sequence of SEQ IDNO:1.
 29. The recombinant DNA molecule of claim 1, wherein therecombinant DNA molecule comprises a nucleotide sequence having at least99% sequence identity to a sequence of SEQ ID NO:2.
 30. The recombinantDNA molecule of claim 1, wherein the recombinant DNA molecule comprisesa nucleotide sequence having at least 99% sequence identity to asequence of SEQ ID NO:3.
 31. The recombinant DNA molecule of claim 1,wherein the recombinant DNA molecule comprises a nucleotide sequencehaving at least 99% sequence identity to a sequence of SEQ ID NO:4. 32.The recombinant DNA molecule of claim 1, wherein the recombinant DNAmolecule comprises a nucleotide sequence having at least 99% sequenceidentity to a sequence of SEQ ID NO:5.
 33. The recombinant DNA moleculeof claim 1, wherein the recombinant DNA molecule comprises a nucleotidesequence having at least 99% sequence identity to a sequence of SEQ IDNO:6.
 34. The recombinant DNA molecule of claim 1, wherein therecombinant DNA molecule comprises a nucleotide sequence having at least99% sequence identity to a sequence of SEQ ID NO:9.
 35. The recombinantDNA molecule of claim 1, wherein the recombinant DNA molecule comprisesa nucleotide sequence having at least 99% sequence identity to asequence of SEQ ID NO:10.